Ghrelin changes food preference in scheduled fed animals

Transcription

1 Ghrelin changes food preference in scheduled fed animals from high fat diet to chow Kim Hellgren Degree project for Master of Science (One Year) in Biology Human Physiology 45 hec Autumn 2014 Department of Biological and Environmental Sciences University of Gothenburg Examiner: Thrandur Björnsson Department of Biological and Environmental Sciences University of Gothenburg Supervisor: Suzanne Dickson Department of Physiology/Endocrinology University of Gothenburg

2 Contents... 2 Abstract... 1 Abbrevations... 2 Introduction... 3 Aim... 6 Materials and Methods... 7 Animals... 7 Breeding and selection of GHS- R1A KO mice... 7 Scheduled feeding and food measurement... 8 ICV cannulation ICV injections Statictical analysis Results Study 1: Rats Study 2: Mice Discussion Conclusion Acknowledgements References:... 25

3 Abstract Obesity is at such a level at that it is referred to as an epidemic with reports of up to 80% of European adults being overweight. Ghrelin is a gut peptide with orexigenic effects that also has been shown to have a connection to the reward system and it s been suggested that highly palatable food increases this reward response. Human eating behaviour goes beyond that of energy homeostatic reason and great emphasis is put on the meal. To mimic this in an animal model this thesis uses a scheduled feeding regime. Scheduled feeding allows the animals to have ad libitum access to a regular diet and with the addition of two hour access to highly palatable food, in this thesis a high fat diet. Previous studies have shown that both rats and mice will rapidly adapt to this regime and binge on the high fat diet as a result. For this thesis two different animal models will be used in two studies. First study will be with intracerebro ventricular cannulated rats that will be injected with ghrelin and a ghrelin antagonist after habituated to the scheduled feeding regime. Second study will be performed with ghrelin receptor deficient mice. A combination of these two studies should tell us of ghrelin s involvement in the binge-like feeding behaviour. With injected ghrelin we see a shift in food preference from high fat diet towards chow intake. The total caloric daily caloric consumption is not significantly altered but an increase of body mass is shown. Knock out mice are able to binge in a similar way to wild type, which indicates that ghrelin is not necessary to induce a binge-like eating behaviour. The conclusion of this study is that we may have misunderstood ghrelin to quite some extent and the needs for further studies are great. 1

4 Abbrevations acsf ANOVA BED Con CPP GHS-R1A HFD ICV JMV KO SEM SF VTA WT ZT cerebro spinal fluid analysis of variance binge-eating disorder control feeding (animal group) conditioned place preference test Growth hormone secretagogue receptor 1A (Ghrelin receptor) high fat diet intracerebro ventricular Ghrelin antagonist knock-out Standard error of the mean Scheduled feeding (animal group) ventral tegmental area wildtype Zeitgeber time 2

5 Introduction Amidst a growing obesity epidemic, there is much need to discover new mechanisms for obesity with a view to new prevention and treatment strategies. The World health organization (WHO) reports that obesity has doubled since In 2007, roughly 30-80% of European men and women were overweight and 150 million adults an 15 million children were expected to be obese in 2010 (World health organization, 2007). In this project we will be exploring the impact of a stomach-derived hormone, ghrelin on behaviors linked to appetite. This research is inspired by studies exploring mechanisms for bariatric (weight loss surgery), as gut hormones such as ghrelin have an altered secretion after this surgery. As we will discuss, ghrelin orchestrates a host of pro-obesity behaviors important for feeding control. It is anticipated that new research focusing on the brain ghrelin system will reveal new information regarding why people become obese, in what way ghrelin may contribute to the weight loss after bariatric surgery and in what way the brain ghrelin system could provide a target for future obesity treatments. With obesity being related to several diseases such as diabetes, hypertension and high serum cholesterol (Paeratakul et al., 2002), it is becoming more and more crucial to work out all possible explanations to the occurring epidemic. It is widely speculated that the reasons for this epidemic are i.e. easily available food (Nelson et al., 2006; Wiecha et al., 2006), sedentary lifestyle (Berkey et al., 2000; Kruger et al., 2006) and genetics (Clement, 1999). But there is no question to the general reason for obesity; it is simply a mismatch between energy consumption and expenditure. Therefore the mechanism and driving force of the extra energy intake becomes of interest. Recent advances have suggested that studies focused on feeding behavior and its control may be required to answer the questions at hand (Hebebrand et al., 2014). The obvious reason for feeding is of course survival, to be able to maintain energy homeostasis. But there is also a hedonically feeding behavior, or a pleasure feeding. As so elegantly put by Saper et al. If feeding were controlled solely by homeostatic mechanisms, most of us would be at our ideal body weight and people would consider feeding like breathing or elimination, a necessary but unexciting part of existence. So does 3

6 the solution lie within the mechanisms of the hedonic feeding behavior? The authors also stated that if presented with highly palatable food, almost any mammal will eat more than that required by energy homeostatic reasons (Saper et al., 2002). However, human feeding behavior is considerably more complex than that of other animals. With feeding and mealtime being influenced by mental, emotional, social and cultural states, human feeding behavior is hard to mimic and quantify. There are also several psychological aspects that have impact on feeding behavior. Anorexia nervosa and bulimia nervosa are ever growing concerns leading to mortality in several cases (Keel et al., 2003). Field trials show that as many as 30% of individuals seeking treatment for obesity, can be diagnosed with binge eating disorder (BED). BED being characterized as bulimia nervosa without the compensatory behavior (such as purging) has shown a significant increase for the second half of the twentieth century (Kendler et al., 1991; Hundson et al., 2007) and may very well a greatly contributing factor to the epidemic. It has been suggested that abnormal eating behaviors affect circulating levels of the orexigenic hormone ghrelin (Tanaka et al., 2002). Ghrelin, a 28 amino acid peptide, was discovered and purified from rat stomach in 1999 (Kojima et al., 1999) and later on cloned in rats and humans. The difference between human and rat ghrelin is just two acid substitutions which suggest that ghrelin has an important physiological role (Wren et al., 2000b). It is produced primarily in the oxyntic glands of the stomach by the X/A-like endocrine cells (Date et al., 2000) but also in the hypothalamic arcuate nucleus (Kojima et al., 1999; Nakazato et al., 2001). It plays an important role in the peripheral, as well as the central regulation of energy balance (King et al., 2011) and has been shown to increase food intake and body weight when administered systemically to rats (Wren et al., 2000a; b; 2001) and increase food intake in humans (Tschöp et al., 2000). Anorexia nervosa and bulimia nervosa patients as well as lean humans show increased plasma ghrelin levels compared to obese humans. It is theorized that the lower ghrelin in obese humans are due to a positive energy balance that is associated with obesity (Tschöp et al., 2001; Korek et al., 2013). Food consumption seems to suppress ghrelin levels in lean but not in obese humans (Ghatei, 2002). 4

7 Receptors for ghrelin (growth hormone secretagogue receptor 1A; GHS-R1A) has been found not only in the hypothalamus, the center of energy homeostatic control, but also in different areas in the hippocampus connected to the reward system, such as the ventral tegmental area (VTA) (Howard et al., 1996; Guan et al., 1997) and in 2005 the results further strengthened by showing an increased food intake after ghrelin injection into the VTA (Naleid et al., 2005). In three different publications Jerlag et al. showed that ghrelin activates the cholinergicdopaminergic reward system via the mesolimbic pathway. They speculate that the reason for this is an extra benefit compared to the cost for activities such as food seeking (Jerlhag et al., 2006; 2007; 2008). They also showed that a ghrelin injection in the VTA resulted in an increase of extracellular accumbal dopamine; at the same time Abizaid et al. showed an increase in dopaminal neuron activity with whole-cell patch clamp recordings (Abizaid et al., 2006). Increased ghrelin levels in alcoholics have also been shown in several cases and have been correlated with increased alcohol cravings and abstinence (Kim et al., 2005; Kraus et al., 2005; Addolorato et al., 2006). This further point towards a close relationship with the reward systems. And interestingly enough, it seems that not only is ghrelin involved in the reward with food intake, but also that the reward, or at least the effect of added ghrelin, is greater with energy-dense foods (Egecioglu et al., 2010). As previously stated, it has been speculated that abnormal eating behavior affects circulating ghrelin levels. Schedule feeding regimes have shown an induced over consumption of palatable food over 2-hour periods in both rats and mice. This beyond that necessitated for energy homeostatic reasons and without any greater reduction of ad libitum feeding on less palatable food choices for the remaining 22 hours of a day (Bake et al., 2014a). This may be the most relevant laboratory induced scenario demonstrating human tendencies to overconsummate fat and sugary foods. Ghrelin s involvement in this kind of scenario is however for the time being not fully understood. 5

8 Aim In this thesis we will seek evidence for the involvement of the central ghrelin signaling system for the regulation of large meals. Binge-like behavior can be induced in rats and mice by presenting palatable food with only a short access time (scheduled feeding), even when the animals are not food restricted (Bake et al., 2013; 2014a). Here, we use a combination of two studies, a pharmacological manipulation in rats and genetically modified mice, to determine the potential role of ghrelin in the modulation of binge-like behavior. In study 1, once rats are habituated to scheduled feeding and display binge-like feeding behavior on palatable food, we will deliver either ghrelin or a ghrelin antagonist, by intracerebro ventricular (ICV) injection into the lateral ventricle, to observe the impact of altered ghrelin signaling on binge-like meal eating. The hypothesis is that binge-like eating behavior seen after the rats have been adapted to the scheduled feeding routine is at least partially mediated by central ghrelin signaling. We expect that the eating behavior will be altered by ghrelin; the food intake of the palatable diet consumed during scheduled feeding will be increased with ghrelin injections and decreased when injecting an antagonist. In a second study using GHS-R1A knock-out (KO) mice we will further investigate the need for ghrelin to initiate a binge-like behavior. Using genetically modified mice that lack the GHS-R1A receptor, we will expose mice to the same scheduled feeding regime used for the rats. Were ghrelin to play a great factor in this behavior no difference would be seen in their eating behavior. The hypothesis is that the GHS-R1A KO mice will not habituate to the scheduled feeding and will instead keep a similar feeding pattern to that of regular ad. libitum feeding. 6

9 Materials and Methods Animals For this thesis, two animal experimental studies were performed, one in rats and the other on in mice. Male Sprague-dawley rats (n=16) were acquired from Charles River, Germany at 7 weeks of age and a body weight of g. They were kept under a 12h:12h light/dark cycle with the onset of light at 20:00 (Zeitgeber Time (ZT) 0) and acclimatized for one week prior to experimental procedures. GHS-R1A KO mice and their respective wild-type (WT) littermates were generated from crosses between heterozygous breeding pairs (see below) from a colony kept at Experimental Biomedicine at University of Gothenburg, originating from AstraZeneca. After weaning at 3 weeks of age they were housed in group cages with their littermates. The mice were kept at a 12h:12h light/dark cycle with onset of light at 06:00 (ZT0). Once they reached 7 weeks of age 25 selected male mice were single housed and transferred into a reversed light/dark cycle with light onset at 16:00 (ZT0) and acclimatized for one week prior to study start. All animals had ad libitum access to chow and water unless otherwise specified. The studies were carried out with ethical permissions from the Animal Ethics committee at the University of Gothenburg and in accordance with legal requirements from the European Community. Ethical permit numbers: (rats), (mice) and (breeding of genetically modified mice). Breeding and selection of GHS-R1A KO mice GHS-R1A KO mice and their respective WT littermates were generated from crosses between heterozygous breeding pairs. After weaning at 3 weeks of age they were group-housed together with all their littermates (KO, WT and heterozygous mice). At the same time the mice were marked by ear punch. The ear punch was used for genotyping purposes Once they reached 7 weeks of age 25 male KO and WT mice were selected for the study. 7

10 Scheduled feeding and food measurement For both animal experimental studies two different diets were used, a standard pellet diet (Harlan labs, Indianapolis, IN, USA; #2016; 22% protein, 66% carbohydrate, 12% fat by energy, 3.00 kcal/g; chow) and one high fat diet (Research Diets, New Brunswick, NJ, USA; #D12492; 20% protein, 20% carbohydrate, 60% fat by energy, 5.24 kcal/g; HFD). Food intake was measured in grams, converted to and presented in kcal. Scheduled feeding (detailed in fig. 1), allowing the animals access to a palatable diet for a limited time period, began at ZT18 with 2 h access to HFD in addition to chow and was started one week after the rats arrival while they were still group housed. Mice were put on the same scheduled feeding regime at 9 weeks of age i.e. one week after single housing. In rats, ICV cannulation surgery (detailed below) was started one week later and the surgery took a total amount of four days before all rats were done. Directly after surgery the animals were moved to single housing where the scheduled feeding continued as earlier. The rats were housed and acclimated to an automated feeding and drinking monitoring system (TSE LabMaster; TSE systems, Bad Homburg, Germany) that digitally measured food consumption by weight. Data was then analyzed using a customized R (R GUI 1.65 Mavericks build (6833), R Core team, Vienna, Austria) script which would check for differences larger than 1 g within a 5 min period concerning the HFD. When such a difference was found, the script would then acquire the value of 1, 2, 4, 6, 18, 24 and 48 hours (for both chow and HFD) following the time-point where the difference was found. Results were printed to a separate file together with the time-point used. This time-point was then matched manually to notes from injection times. If any deviation was found the values would be acquired manually. Scheduled feeding was carried out for a total of six weeks with three weeks during which injections were performed. The mice were left to acclimatize to single housing and the reversed light cycle for two weeks and where then divided in to four groups. Group 1 consisted KO mice that had 2 h access to 8

11 HFD between ZT18 and ZT20 in addition to chow (KO SF, n=7) as did group 2 that consisted of WT mice (WT SF, n=6). Group 3 (KO con, n=6) and 4 (WT con, n=6) were used as control groups and only had access to ad libitum chow. The food was however measured at the same time (ZT18 and ZT20) to account for the disturbance that was caused to the mice in group 1 and 2. In both studies, body weights were recorded at frequent intervals, e.g. three times a week or prior injection and 24 h and 48 h after injection and great consideration to the three r s and the animals welfare was taken throughout the duration of the study. ZT0 Injection (only rats) Access to HFD Scheduled feeding Food measurement time- points are indicated with: Injection day 1 day after injection 2 days after injection Figure 1: A detailed schedule for the different time-points regarding the scheduled feeding. Each line on the bar symbolizes one hour of the day. 9

12 ICV cannulation For the rat study, an IVC cannulation was performed under anesthesia generated by a Ketalar, Rompun mixture (3:1, conc: 2 ml kg -1 ). The calculated dose was injected intra-peritoneal (IP) to assure the animal was kept under heavy sedation throughout the entire duration of the procedure. The fur of an area from the flat of the nose, back between the ears and down the neck was shaved using electric clippers. Rats were then fastened in a stereotaxic frame (Model 942, David Kopf Instruments, Tujunga, CA, USA). A cut along the midline was made using a scalpel to expose the skull. The surface was then relieved of fascia and cleaned from blood using cotton tips. Bregma was located and considered origin for lateral and posterior coordinates, as zero point for depth the surface of the skull bone itself were used. Coordinates for the lateral ventricles (-0.9 mm posterior, ±1.6 mm lateral and -2.5 mm ventral relative to bregma) were taken from the Rat Brain Atlas(Paxinos and Watson, 2009). Regarding the lateral placement both +1.6 mm and 1.6 mm were used in a 1:1 ratio to allow for injections in both right and left lateral ventricle. An hole was made using a handheld drill at the coordinates specified and a single guide cannula (Bilaney, Kent, UK; #C313G, 4 mm) was inserted to the depth specified. Four additional smaller holes, evenly distributed over the surface (one anterior to bregma, one posterior to lambda, one left to the midline and one right to the midline), were made using the hand held drill and fitted with stainless steel screws (Agnthos, Lidingö, Sweden; #MCS1x2) which were placed in the four holes to work as anchor points for the dental cement (Agnthos, Lidingö, Sweden; #7508, #7509). The whole surface was then covered with dental cement, extra care given to assure that surface was completely dry and that the dental cement would go under the screws, and hold the guide cannula in place without interfering with the thread on the cannula. A dummy cannula (Bilaney, Kent, UK; #C313DC) was inserted into the guide cannula to prevent obstruction. All rats for allowed to recover for one week. Cannula placement was controlled with a 2 µl angiotensin II (10 ng µl -1 ) injection, placement was considered correct if the rat drank from its water bottle within 5 minutes and also drank more than 5 ml during a 30 minute period following the injection. The test was first done with 2 mm injectors (Bilaney, Kent, UK; 10

13 #C313I, 4mm), and the rats that did not qualify with the 2 mm were tested the next day with 2.5 mm (Bilaney, Kent, UK; #C313I, 4mm) where all the rats passed. ICV injections Injections were administered using microliter syringes connected to a catheter which were fitted with an ICV injector (Bilaney, Kent, UK; #C313I, 4mm) of previously decided length for each individual (acquired by the angiotensin II test) since the exact length would depend on the placement of the cannula. Hormones used were ghrelin (Tocris, Bristol, UK; #1463) and the GHSR1 antagonist JMV2959 (JMV; AEterna Zentaris, Frankfurt, Germany; #D ) both diluted in artificial cerebro spinal fluid (acsf; Tocris, Bristol, UK; #3525). Injections were made in a counterbalanced manner to be able to minimize the amount of animals used. In other words, at the end of the experiment all rats received each substance and concentration at least once. Ghrelin as well as JMV were injected in two different doses, ghrelin 1 µg and 2 µg as well as JMV 8 µg and 12 µg both similar to concentrations earlier used by Alvarez-Crespo et. al (Alvarez-Crespo et al., 2012) all diluted with acsf to a total injection volume of 2µl. As a control 2 µl acsf was injected. One rat lost its cannula at the end of the study which decreased n from 16 to 15 for fasting and JMV 8 µg results. Injections were performed just before start of scheduled feeding (ZT18) and a minimum of 48 hours were always left between injections. Food consumption was measured at a total of 7 time points after injection, 1, 2, 4, 6, 18, 24 and 48 hours (see fig. 1). Bodyweights were recorded at the time of the injection, 24 and 48 hours later. To allow comparison with natural hunger, the animals were fasted for 16 h at the end of the study and food intake was measured for 24 h post fasting. 11

14 Group housed scheduled feeding Each rat was single housed after its surgery Ghrelin injections One week ICV surgery Angiotensin II JMV injections Figure 2: The figure show a detailed schedule of the study with each Monday represented with a long line and the subsequent days with a shorter line. Statictical analysis All statistical analysis was done using R (R GUI 1.65 Mavericks build (6833), R Core team, Vienna, Austria). In the rat study, when data was found not to be normally distributed (Shaprio-Wilk test < 0.05) and/or showed heterogeneity (Levene s test < 0.05) consumption data was analyzed using Kruskal-Wallis rank sum test and pairwise comparisons using Wilcoxon rank sum test with bonferroni as p-value adjustment method. Otherwise a one-way analysis of variance (ANOVA) with a Tukey HSD post hoc test performed. Statistical analysis was made between all treatments within each time point using cumulative values of kcal for the different time points. Analysis were made separately for HFD and chow but also combined. 12

15 In the mouse study, data were analysed by ANOVA for effect of genotype (WT vs. KO) and feeding regime (scheduled feeding vs. control feeding) and for interavtion between these factors. Post-hoc and planned comparision were assessed with Tukey HSD post hoc test. Significance was considered at p < 0.05 for all data. The data are presented as mean ± standard error of the mean (SEM). 13

16 Results Study 1: Rats Food consumption When schedule fed, the rats showed a binge-like feeding behavior. During control condition (injection of acsf), the rats would consume nearly 60 kcal within two hours and almost 50 kcal of those already in the first hour (fig. 3). This accounts for 60% of their daily caloric consumption (fig. 7) and is consisted solely of the HFD (fig. 4). 48 hours after control injections the rats would gain an average of 3 g and have eaten a total of 190 kcal (fig. 5), out of which almost 120 kcal would be acquired from the two instances of scheduled feeding and HFD that occurs within 48 h (fig. 4 & 5). Injections of 1 µg ghrelin significantly decreases HFD intake during the 2 hours (p = 0.02; fig. 3) and increase the amount of chow consumed for up to 24 hours (fig. 4). The higher concentration of ghrelin (2 µg) showed no significant changes (p = 0.14) but did show an increased chow consumption for up to 48 hours (fig. 4 & 5). The percentage of the daily caloric consumption from HFD was lowered to about 40% (fig. 6) but does not affect total caloric intake for 24 or 48 hours (fig. 5). Body weight however, increases between 6 and 7 grams to a total increase of approximately 10 g, 48 hours post injection (fig. 7). Fasting the rats for 16 hours led to a similar increase in chow consumption for the 24 hours measured (fig. 4) including an increase during scheduled feeding. It did not show any change in HFD consumption during the scheduled feeding compared to control injections but did significantly increase HFD intake compared to both ghrelin concentrations (p = for 1 µg and p=0.01 for 2 µg; fig. 3). This then lead to a significant increase in total caloric consumption for all time-points up to 24 hours (6 h, p = 0.006; 18 h, p = 0.02; 24 h, p = 0.02; fig. 5) compared to control. 14

17 Injections using JMV 8 µg or 12 µg did not reveal significant effects in any of the measured parameters. A B Figure 3: Cumulative energy intake from HFD during schedule feeding after injection of Ghrelin or JMV. (A) Cumulative HFD consumption is significantly decreased by ghrelin injections of 1 µg for both time points (marked with an asterix, p<0.05). Injections with ghrelin of both concentrations significantly lowers HFD consumption compared to fasting (p<0.05, marked with a hash). (B) HFD consumption stays unaltered with JMV injections. Values are presented as mean kcal consumption with error bars representing ±1SEM, n=16 for all but fasting and JMV 8 µg where n=15. A B Figure 4: Cumulative energy intake from chow over 48 h after injection of Ghrelin or JMV (A) Ghrelin injections, as well as 16h fasting, significantly increases chow intake for up to 24h compared to acsf injections. The higher concentration of ghrelin (2 µg) shows a significant increase for up to 48 h compared to the lower concentration (1µg) which only is significantly differed from control for 6 h. (B) JMV fails to significantly alter chow intake. Values are presented as mean kcal consumption with error bars representing ±1SEM, n=16 for all but fasting and JMV 8 µg where n=15. Asterisks indicates significant difference from control (p<0.05) 15

18 A B Figure 5: Cumulative energy intake from both HFD and chow over 48h after injection of Ghrelin or JMV. (A) The total amount consumed (chow + HFD) is not altered by ghrelin injection but is increased after 16 hour fasting (hash symbol show significant difference from fasting, p<0,05). (B) JMV injection does not show any effect on total caloric intake. Values are presented as mean kcal of HFD and chow consumption combined with error bars representing ±1SEM, n=16 for all but fasting and JMV 8 µg where n=15. Figure 6: Percentage of caloric intake from chow and HFD after injections with ghrelin. Ghrelin injections of both concentrations significantly decreases the amount of daily caloric intake consisting of HFD. Values are presented as mean percentage of total kcal consumed with error bars representing ±1SEM, n=16. Asterisks indicates significant difference from control (p<0.05). 16

19 A B Figure 7: Average weight gain over 48 hours after injection of Ghrelin or JMV. (A) Ghrelin injections of both concentrations significantly increase weight gain 48 hours after injection compared to acsf injections. (B) No significant changes with JMV injections. Values are presented as mean weight gain in grams with error bars representing ±1SEM, n=16. Asterisks indicates significant difference from control (p<0.05). Study 2: Mice When schedule fed the mice will increase change their feeding pattern to indulge on HFD for the two hours it s available and decrease their chow. Both KO mice and WT will significantly increase their HFD intake as soon as they habituated to the regime (fig. 8A; p< for KO and WT). There is however no significant difference between KO and WT at day 18 (p=0,25). The scheduled fed mice will also significantly decrease their chow intake during the 22 h where they are not schedule fed (fig. 8B; p<0.0001). No significant changes are seen in either total caloric intake (fig. 8C) or bodyweight gain (fig. 8d) 17

20 A B C D Figure 8: Food consumption for mice and BW changes (A) A significant increase in HFD intake, during the scheduled feeding, is seen with habituation but no significant differences between KO and WT mice. (B) Both KO and WT mice reduce their chow intake for the remaining 22 hours compared to the ad libitum fed mice, no differences were seen between KO and WT. (C) No significant changes for the total daily caloric intake is seen. (D) There are no significant changes in bodyweight between the groups. Values are presented as mean in kcal or grams with error bars representing ±1SEM, n=7 for KO-SF and n=6 for the other groups. Asterisks indicates significant difference from hashes (p<0.05). 18

21 Discussion Binge-eating disorder (BED) is a condition that affects the society with an ever increasing body mass worldwide (Kendler et al., 1991; Hundson et al., 2007; World health organization, 2007). Indeed, binge eating obesity is a term now coined to describe a subset of obese patients that have become obese due to binge behavior for foods. The physiological mechanisms behind it are currently very poorly understood. We need a better understanding of these mechanisms if we are develop new therapeutic strategies to treat this disorder. Given the overlapping neurobiology between binge-eating disorders and other eating disorders such as bulimia nervosa, it remains possible that such therapies may also have benefit for eating disorders more generally. When trying understand mechanisms underlying binge-eating it is important to find a good animal models that simulate human behaviour. A very simple model for this is scheduled feeding in which rodents are given access to highly palatable food (e.g. high fat diet, HFD) for only a short period of time (typically 2 hrs) over and above their regular chow. The animals rapidly adapt their feeding behavior and show hyperphagia during the time when the palatable diet is available (Berner et al., 2012; Bake et al., 2014b). The newly adapted eating behavior shows some characteristics of binge-eating obesity and can thereby be studied to try and find a physiological controller of this behavior. Here we sought to determine whether the central ghrelin signaling system has a role in bingelike eating behavior. We performed HFD schedule feeding studies in (i) rats treated with ghrelin or a ghrelin antagonist administered by direct injection to the brain ventricles and also (ii) genetically modified mice that lack the ghrelin receptor. We also measured total food intake per se as, based on previous studies (Wren et al., 2001), it was expected that brain delivery of ghrelin would increase food intake in rats whereas a ghrelin antagonist would decrease it. Due to the lack of ghrelin receptors in the KO mice, we anticipated that they would not change their behavior when schedule fed. 19

22 Remarkably, we found that ICV. delivery of ghrelin decreased consumption of HFD during the scheduled feeding (binge) period and instead preference redirected towards increased chow diet (figs. 3A and 4A). The overall caloric consumption is not changed with ghrelin injections (fig. 5A) but the weight gain was affected (fig. 7A). We were able to replicate the change in food choice seen during the two hours of scheduled feeding by fasting the rats for 16 hours. However, unlike the ghrelin-treated rats, the fasted rats did not decrease their HFD intake. This finding, that ghrelin and fasting impact differently on food intake during a scheduled feed of HFD is unexpected as ghrelin is thought to mediate, at least in part, many of the effects of fasting on food intake. The KO mice show the same trends as the control i.e. are able to show a binge-like feeding behavior even with the lack of ghrelin receptors (fig. 8). We are able to show changes in food choice during the scheduled feeding by injecting ghrelin. These changes however are not in accord with our hypothesis. Instead of an increase in HFD we found instead a shift towards chow intake which leads us to conclude that ghrelin is not a driving force behind the choice of palatable diets, at least not in a binge-like eating behavior. This to the contrary of results shown by Shimbara et. al (Shimbara et al., 2004) that, using a conditioned place preference test (CPP), in which ghrelin increased preference for a compartment previous paired to the HFD. Another interesting point is that in schedule fed rats, ghrelin appears to lose its overall orexigenic effect. We found no increase in overall food intake using 2 different doses of ghrelin, whereas in free feeding rats, ghrelin is orexigenic in both rats (Wren et al., 2000b) (Kojima et al., 1999)and human subjects(wren et al., 2001). We may infer that ghrelin is unlikely to be a primary drive for the over consumption of caloric foods in individuals suffering from binge-eating obesity. Consistent with this the studies in the ghrelin receptor knockout mice did not suggest a role for the central ghrelin signaling in binge-like behavior for food in the schedule feeding paradigm. Lack of effects in the ghrelin receptor knockout mice is interpreted to indicate that the receptor is not needed for binge-like behavior for food. It should be pointed out, however, that nature has introduced a lot of redundancy in feeding pathways because they are so essential to 20

23 life. It may be that some other pathways can compensate for the lack of the ghrelin signal in these mice. We expected the ghrelin antagonist, JMV to decrease their HFD intake during the binge period and that total intake would be lowered. Unfortunately we saw no changes at all with the JMV injections. It is difficult to speculate about the reason for this. Previous studies have shown decreases in food intake with JMV (Egecioglu et al., 2010). We cannot rule out the possibility that the JMV compound lacks biological activity (eg a faulty batch of the compound) in this study and so we are reluctant to draw too many conclusions from the studies with this antagonist. The results suggest that central ghrelin signaling can influence the choice of foods in rats exposed to a scheduled feeding paradigm. During scheduled feeding, control rats eat almost no chow and indulge on the HFD, whereas ghrelin injected animals start to eat chow. Thus, in a situation when rats normally eat a lot of HFD, ghrelin is able to redirect the food choice towards a less caloric food. Tentatively, this could suggest a novel role for ghrelin, to promote healthier food choice (chow>hfd). Consistent with this, some preliminary data from another member of Prof Dickson s group has found that, in rats fed a choice diet of sugar, lard or chow over 2 weeks, acute brain ghrelin injection redirects the choice towards chow. However, the physical properties of the two different diets (HFD versus chow) should be taken into consideration. While the HFD is palatable and energy-dense, it is also somewhat hydrophobic and would not absorb liquids or swell in size when in contact with e.g. water. By contrast, the chow soaks up water and expand greatly when kept moist. This may play a role in the satiation effect of the diet. Tentatively, it may be that an extreme hunger sensation would drive the rats to seek out the chow solely for its satiation properties. This theory could also be supported by the fact that fasting shows divergence with the ghrelin injections with respect to their dietary choice during the binge period. The increase could be explained by the fact that the stomach in the case of fasting actually is empty while the injected rats still had chow available ad libitum. It shall not go unmentioned though that fasting also increases 21

24 ghrelin levels which complicates the possibility to draw a clear line between the treatments and the effects seen. Further studies are needed to address ghrelin s role in food choice paradigms. Suggestions for future studies would be an ad libitum food choice study in combination with injections of ghrelin and an antagonist. It may also be an idea use operant boxes in a similar fashion Shimbara et. al (Shimbara et al., 2004) with the modification of using 2 levers delivering different diets and then see if the preference can be altered using pharmacological injections of ghrelin and ghrelin antagonist. With operant boxes a rat could be trained to choose one of two levers and depending on which lever is chosen it would receive a chow or a sucrose pellet. 22

25 Conclusion We set out to test the hypothesis that ghrelin injected rats would increase their overall caloric intake during scheduled feeding periods and expected that the increase would be due to increased fat consumption. However, strikingly at a time when rats normally binge on HFD, under the influence of ghrelin, they start to eat more chow. The effect of injected ghrelin is that the preference shifts from a highly palatable unhealthy diet to the healthier chow pellet. This leads us in a different direction than many previous studies where HFD diet is considered a greater reward which would be regulated by ghrelin. Whether this means that the ghrelin is not as rewarding as expected or that the reward itself lie elsewhere, cannot be concluded by the present study. As speculated earlier, the chow may contribute to a greater satiation in which case the reward may lie in the satiety as such rather than the caloric intake. In any case, with the results from the current study we may have to reevaluate ghrelin s role in the human feeding pattern and try to look at this from another perspective. 23

26 Acknowledgements My deepest gratitude goes to Dr. Tina Bake who has guided me through this entire endeavor with invaluable insight and support. I would also like to extend my most sincere thanks to Prof. Suzanne Dickson without whom there would have been no study at all. Additionally I would like to thank Carina Babaei Soroori and Robert Jakubowicz for all their help in the lab, Kaisa Askevik for listening to all my confused thoughts and also the entire staff at the Laboratory for Experimental Biomedicine for taking care of our rats and mice throughout the course of the study. Finally I would like to thank our financial contributors EC project Nudge-it. 24

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