The Acute Effects of Olanzapine on Ghrelin Secretion, CCK Sensitivity, Meal Size, Locomotor Activity and Body Temperature

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International Journal of Obesity (2012) 36, 254–261 & 2012 Macmillan Publishers Limited All rights reserved 0307-0565/12 www.nature.com/ijo ORIGINAL ARTICLE The acute effects of olanzapine on ghrelin secretion, CCK sensitivity, meal size, locomotor activity and body temperature

EM van der Zwaal1, M Merkestein1, YK Lam1, MAD Brans1, MCM Luijendijk1, LIH Bok2, ER Verheij2, SE la Fleur1,3,4 and RAH Adan1,4

1Rudolf Magnus Institute of Neuroscience, Department of Neuroscience and Pharmacology, University Medical Center Utrecht, Utrecht, The Netherlands; 2TNO Quality of Life, Zeist, The Netherlands and 3Department of Endocrinology and Metabolism, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

Objective: Significant weight gain is a problematic side effect of treatment with the antipsychotic drug olanzapine (OLA). Previous studies in rats suggest that one of the contributing factors is an impairment in satiation that results in increased food intake. However, the mechanisms underlying this impairment in satiation remain largely unclear. Methods and results: In this study, we determined the effect of OLA on levels of leptin, insulin, ghrelin, cholecystokinin (CCK), glucagon-like peptide-1, peptide YY and amylin in male rats that had received a fixed amount of food. OLA did not affect the secretion of any of these hormones, except for ghrelin levels, which were increased compared with controls. Furthermore, when ghrelin levels were determined in rats just before they received their meal, OLA caused a significant increase in ghrelin levels compared with controls, whereas OLA failed to affect baseline ghrelin levels. Next, we investigated the effect of OLA on the efficacy of CCK to reduce meal size. With coadministration, OLA pretreatment counteracted the reduction in meal size by CCK, although there was no significant interaction between the treatments. Finally, telemetry measurements revealed that acute OLA treatment causes a temporary decrease in both locomotor activity and body core temperature. Conclusion: Taken together, this study shows that acute injection of OLA selectively increases meal-related ghrelin secretion and this may partially underlie the impairment in satiation by OLA. International Journal of Obesity (2012) 36, 254–261; doi:10.1038/ijo.2011.97; published online 10 May 2011

Keywords: food intake; meal size; olanzapine; CCK; ghrelin

Introduction several metabolic side effects and significant weight gain is especially observed in patients treated with OLA.5,6 The introduction of second-generation antipsychotic drugs, The mechanisms underlying antipsychotic-induced such as olanzapine (OLA), has significantly improved the weight gain remain largely unclear. However, several clinical treatment outcome of several psychiatric illnesses, including studies suggest that altered eating behavior is an important schizophrenia and bipolar disorder.1–4 OLA is one of the contributing factor: patients treated with atypical antipsy- drugs considered superior to conventional antipsychotics, chotics such as OLA frequently report an increase in because of reduced extrapyramidal side effects as well as appetite, appear more susceptible to hunger and experience improvement of negative symptoms.1,2 Unfortunately, ingested food as less satiating.7–10 The regulation of appetite second-generation antipsychotics have been associated with and eating behavior is determined by a complex interaction of different systems. The brain receives information about adiposity levels through circulating factors, such as insulin and leptin, and signals from the gastro-intestinal tract Correspondence: Professor Dr RAH Adan, Rudolf Magnus Institute of Neuroscience, Department of Neuroscience and Pharmacology, University through vagal afferents and gut-derived hormones, such as Medical Center Utrecht, Universiteitsweg 100, 3584 GC Utrecht, The ghrelin, cholecystokinin (CCK), peptide YY (PYY) and Netherlands. glucagon-like peptide-1 (GLP-1). These signals are integrated E-mail: [email protected] with input from brain areas that convey the sensory and 4These authors contributed equally to this work. Received 13 January 2011; revised 29 March 2011; accepted 4 April 2011; rewarding aspects of food, and ultimately result in either 11,12 published online 10 May 2011 initiation or termination of a meal. Which components Olanzapine’s effects on meal-related ghrelin and CCK EM van der Zwaal et al 255 of this process are influenced by antipsychotic drugs such as humidity-controlled room (21±2 1C) under a 12/12 h light/ OLA remains largely unclear. dark cycle (lights on at 0300 h). All experimental procedures Several rodent studies, including our own, suggest that were approved by the Committee for Animal Experimenta- OLA affects food intake by impairing the satiation process, tion of Utrecht University and were conducted in agreement which manifests itself by an increase in meal size or short- with Dutch laws (Wet op de Dierproeven, 1996) and term food intake.13–16 This impaired satiation may be due to European regulations (Guideline 86/609/EEC). Rats had free an effect of OLA on the secretion of satiety and/or satiation access to standard lab chow (CRM(E), Special Diet Services, factors, but it could also be due to a reduction in sensitivity Witham, UK) and tap water, except on test days, when food to these factors. was removed as stated below. To investigate whether OLA influences food intake through effects on the secretion of gastro-intestinal peptides that are released in response to a meal, we determined the Surgery effects of an acute injection of OLA on levels of several A week after arrival, rats used in experiment 2 received a hormones that are known to have a role in food intake, transmitter (TA10TA-F40, Data Science International, St Paul, namely leptin, insulin, ghrelin, CCK, PYY, GLP-1 and MN, USA) in the abdominal cavity, to continuously monitor amylin.12 This revealed an increase in periprandial levels of body core temperature and locomotor activity. Surgery was the orexigenic hormone ghrelin in OLA-treated rats. performed under fentanyl/fluanisone (Hypnorm, Janssen Administration of ghrelin directly into the brain has Pharmaceutica, Beerse, Belgium, 0.1 ml per 100 g intra- previously been associated with a more marked increase in muscularly) and midazolam (Dormicum, Roche, Woerden, meal frequency than in meal size.17,18 This seems to be in The Netherlands, 0.05 ml per 100 g intraperitoneally (i.p.)) contrast with the meal patterns of olanzapine-treated rats, anesthesia. In addition, an i.p. canula was implanted to which exhibit an increase in meal size accompanied by a administer drugs painlessly. This canula consisted of a silicon compensatory decrease in meal frequency.14,16 However, tubing (S0.6 Â 1.2D, Raumedic, Helmbrechts, Germany), peripheral administration of exogenous ghrelin prior to a B20 cm in length, of which the proximal end was positioned buffet-style meal has been shown to increase subsequent intra-abdominally. The intra-abdominal part consisted of caloric intake in humans.19 Furthermore, peripherally approximately 4 cm of tubing that had been perforated at administered ghrelin affects meal size in rodents as well, regular intervals to prevent blockage. The distal end was depending on the experimental circumstances.20,21 This tunneled subcutaneously (s.c.) and attached to a connector effect on meal size may be explained by the finding that pedestal (313-000, Plastics One, Roanoke, VA, USA) that was pretreatment with ghrelin was able to completely abolish the fixed to the skull using dental cement and screws. Immedia- effect of CCK on food intake, an effect that seems to be due tely after placement of the canula, it was flushed with saline to ghrelin preventing the activation of vagal nerves by (1 ml kgÀ1) to remove any debris and a dust cap placed on the subsequent CCK administration.22 As the meal patterns of tip of the connector pedestal. Additionally, cannulas were OLA-treated rats markedly resemble those of rodents lacking flushed with saline on the first postoperative day, and at the CCK-1 receptor23 and ghrelin has a role in CCK least twice weekly throughout the experiment to prevent sensitivity,22 we hypothesized that OLA might reduce the blockage. Carprofen (Rimadyl, Pfizer Animal Health, Capelle sensitivity to endogenously released CCK in response to a a/d IJssel, The Netherlands, 0.5 mg per 100 g s.c.) was meal. administered as pain medication preoperatively and once To test this hypothesis, we investigated the effect of daily postoperatively for 2 days. Animals were allowed to OLA pretreatment on the efficacy of CCK to reduce meal recover for 2 weeks before the first drug administration. size in male rats. In this behavioral paradigm, OLA was administered acutely to enable repeated measurements in individual animals. In our previous study, OLA was Drugs administered continuously and it affected not only meal OLA (Chempacific Corp., Baltimore, MD, USA) was size but also locomotor activity and body core tempera- dissolved in a minimum quantity of 1 M hydrochloric acid 16 ture. Therefore, these parameters were also monitored in and diluted with saline. If necessary, 1 M sodium hydroxide the present study, in order to compare the results of acute was used to adjust the pH to B5.5. A stock solution of administration with previous findings. 4mgmlÀ1 was made at the start of the experiment, this solution was aliquoted, stored at À20 1C and used during the experiment to prepare fresh solutions by diluting with Materials and methods saline as needed. Similarly, a stock solution (50 mgmlÀ1) of CCK octapeptide Animals (sulfated) ammonium salt (Bachem AG, Bubendorf, Male Wistar rats, weighing 275–300 g, were purchased from Switzerland) was prepared using phosphate buffered saline Charles River Laboratories (Crl-Wu, Sulzfeld, Germany). (PBS), stored in aliquots at À20 1C and diluted with PBS on They were individually housed in a temperature and test days.

International Journal of Obesity Olanzapine’s effects on meal-related ghrelin and CCK EM van der Zwaal et al 256 The dose of OLA was chosen based on the fact that acute phase. On the day of treatment, food was removed at ZT4 administration of 1 mg kgÀ1 OLA in rats results in peak and rats received an i.p. injection of saline (n ¼ 5) or receptor occupancy levels that are comparable to humans.24 1mgkgÀ1 OLA (n ¼ 6) between ZT6 and ZT7. They were The dose of CCK was chosen based on a pilot experiment killed by decapitation 45 min after drug administration. that indicated this was the minimum effective dose to reduce meal size in our paradigm. Collection of blood and tissues Experimental designs After rats were killed by decapitation, trunk blood was collected and immediately placed on ice. For experiment 1A, Experiment 1 tubes collecting samples for CCK measurement contained To determine the effects of acute OLA administration on the approximately 12 mmol of EDTA and 75 mg aprotinine per ml secretion of different meal-related hormones, we used three blood. Tubes used to collect samples for other hormone À1 different setups. In each case, rats had been used in a different measurements contained approximately 12 mmol ml À1 À1 set of acute pharmacological experiments previously, but had of EDTA, 75 mgml of aprotinine and 10 mlml DPP IV- gone through a wash-out period of at least 2 weeks. During inhibitor (Millipore, Remecula, CA, USA). For experiment 1B this time, food intake and body weight gain were also and 1C, lithium–heparin-containing tubes were used with monitored so that, for each experiment, treatment groups approximately 12 mmol of EDTA and 75 mg aprotinine per ml could be matched for both food intake and body weight. of blood. After centrifugation, plasma was stored at À20 1C until further analysis. To confirm that adiposity levels were equal between treatment groups of each experiment, Experiment 1A: Effect on postprandial hormone levels epididymal and s.c. fat pads were dissected and weighed, In this experiment, we investigated the effects of OLA on the and leptin levels determined in all plasma samples. secretion of several circulating satiety/satiation factors in response to a meal. OLA was administered to rats that received a fixed amount of chow at the end of the light Experiment 2: Effect on CCK sensitivity phase. To ensure that all rats consumed the same amount of In this experiment, we examined the effects of OLA on CCK food within a similar time-window, rats were trained in the sensitivity in 23 rats equipped with an i.p. cannula. On test week before treatment: each day, food was removed for the days, access to food was blocked during the last 6 h of the light last 6 h of the light phase, at ZT11 a small portion of chow phase and olanzapine (OLA) (1 mg kgÀ1)orsalinewas was placed in the animal’s homecage and food hoppers were administered via the i.p. cannula between 45 and 30 min returned at the start of the dark phase (ZT12). On the day of before onset of the dark phase. Half an hour later this was treatment, food was removed at ZT6 and i.p. injections of followed by administration of CCK-8 (3 mgkgÀ1)orPBS. saline (n ¼ 7) and 1 mg kgÀ1 OLA (n ¼ 7) were administered Access to food was restored 5 min after a rat had received its between ZT10 and ZT11. Half an hour after injection, rats second injection, which was always within the last 15 min of were offered 2.3 g of chow, which they all consumed the light period. The size of the first meal ingested after food immediately. They were killed 45 min later (75 min after was returned was used to determine the effects of drug injection). administration. Each rat received four combinations of injections in a Latin square design (saline/PBS, OLA/PBS, Experiment 1B: Effect on preprandial hormone levels saline/CCK and OLA/CCK) and served as its own control, In this experiment, we investigated the effect of OLA on allowing a minimum of 2 days between test days. Unfortu- secretion of ghrelin just prior to a meal by determining its nately, not all i.p. canulas maintained proper function during effect just before the dark phase when rats normally initiate the experiment. In these cases, administration of each drug 25 feeding and ghrelin levels are high. The rats used in this combination was performed using regular i.p. injections. experiment had previously been used in a study similar to experiment 2. Therefore, these rats had repeatedly experi- enced food deprivation for the last 6 h of the light phase, Plasma analysis with food being replaced at the start of the dark phase. The serine-3-acetylated form of ghrelin is considered to be On the day of treatment, food was removed at ZT5 and rats responsible for the majority of its biological activities and is À1 received an i.p. injection of saline (n ¼ 7) or 1 mg kg OLA often referred to as ‘active ghrelin’.26 However, the total (n ¼ 8) between ZT10 and ZT11. Rats were killed 45 min after amount of ghrelin present in the circulation consists largely drug administration, in the hour before the beginning of the of the unacetylated form.27 In the present study, effects of dark phase. OLA on levels of both active and total ghrelin were determined. Experiment 1C: Effect on baseline hormone levels Levels of insulin, active ghrelin, GLP-1 (active), PYY (total) In this experiment, we determined effects on basal ghrelin and amylin (active) were measured using a commercially levels, by treating rats with OLA in the middle of the light available luminex kit (Milliplex rat gut hormone panel kit,

International Journal of Obesity Olanzapine’s effects on meal-related ghrelin and CCK EM van der Zwaal et al 257 Millipore) for Luminex X200, according to the manufac- To determine the effects of OLA on locomotor activity and turer’s instructions. Commercially available radioimmunoas- body temperature, a repeated-measures three-way ANOVA say (RIA) kits were used to determine levels of CCK-8 was performed for 24 h after injection, with both drugs and (Euria-CCK, Eurodiagnostica, Malmo¨, Sweden), leptin (Linco time as separate within-subjects factors. This was followed by Research, St Charles, MO, USA) and total ghrelin (Phoenix two-way ANOVAs for different time points, where appro- Pharmaceuticals, Burlingame, CA, USA) according to the priate. If the condition of sphericity was not met, Green- manufacturer’s instructions. For total ghrelin measurement, house–Geisser correction was used. Findings were considered plasma samples were diluted to half of the original statistically significant if Po0.05 (two-tailed). concentration in order to obtain levels in the optimal range for the RIA kit. Obtained values were then multiplied by 2 to calculate ghrelin levels of the original plasma samples. Results Analysis of all samples was performed in duplo, except for CCK-8, because the amount of required plasma was too high. Experiment 1 Table 1 shows plasma concentrations of gastro-intestinal hormones that were determined in OLA- and saline-treated rats after they had received a fixed amount of chow at the end Data analysis of the light phase (experiment 1A). No significant effects were Body weight, food and water intake were measured daily. found on postprandial levels of CCK, GLP-1, PYY and insulin, Meal patterns were determined using data collected by Scales although levels of amylin tended to be increased (Z ¼À1.85, (Department of Biomedical Engineering, UMC Utrecht, The P ¼ 0.064). Furthermore, postprandial levels of active ghrelin Netherlands), a program that records the weight of food were B88% higher in OLA-treated rats compared with hoppers in the home cage automatically every 12 s. A meal controls (Z ¼À1.98, P ¼ 0.048). Levels of total ghrelin after a was defined as an episode of food intake with a minimal meal also tended to be increased by B10% in OLA-treated rats consumption of 1 kcal (0.3 g chow) and a minimal intermeal (Table 2; Z ¼À1.72, P ¼ 0.083). interval of 5 min.16 The intra-abdominal transmitters sent When ghrelin levels were measured in rats just prior to digitized data of body core temperature (1C) and locomotor receiving their food at the end of the light phase (experiment activity (arbitrary units) to a receiver plate present below the 1B), levels of active ghrelin in OLA-treated rats were home cage via radio frequency signals. These data were significantly higher (B104%) than in controls (Table 2; automatically recorded every 10 min using DSI software Z ¼À2.32, P ¼ 0.02). Levels of total ghrelin were also higher (Data Science International, St Paul, MN, USA) and averaged (B27%) compared with controls (Table 2; Z ¼À2.31, per hour for statistical analysis. P ¼ 0.021). In contrast, when OLA was administered in the Statistical analysis was performed using SPSS15.0 (Windows). For plasma levels of different hormones in Table 1 Effect of olanzapine on plasma concentrations of several gastro- experiment 1, Mann–Whitney tests were used to determine intestinal hormones in rats that received a fixed amount of chow (experiment statistical significance. To determine statistical significance 1A) between the effects of different treatment combinations on Saline Olanzapine meal size in experiment 2, repeated-measures analysis of variance (ANOVA) was performed, followed, where appro- Active ghrelin 6.93 (1.81) 13.0 (2.06)* CCK 0.53 (0.16) 0.34 (0.06) priate, by paired samples t-tests. Planned pairwise compar- GLP1 113.2 (14.7) 128.1 (17.3) isons were saline/PBS vs all three treatment combinations PYY 79.9 (8.96) 102.6 (10.5) and OLA/PBS vs OLA/CCK treatment. To determine the main Insulin 3.9 (0.49) 4.5 (0.49) effects of drug treatment, as well as interaction effects, a Amylin 187 (42) 309.9 (38.3) repeated-measures two-way ANOVA was performed, with Abbreviations: CCK, cholecystokinin; GLP1, glucagon-like peptide-1; each drug as a separate within-subjects factor. PYY, peptide YY. *Po0.05. Data are expressed in pg mlÀ1 as mean (s.e.m.).

Table 2 Effect of olanzapine on plasma concentration of ghrelin and leptin levels

Experiment 1A (ZT13) Experiment 1B (ZT12) Experiment 1C (ZT7)

After feeding Before feeding Light phase/baseline

Saline Olanzapine Saline Olanzapine Saline Olanzapine

Leptin (ng mlÀ1) 10.36 (1.11) 9.00 (1.23) 4.44 (0.36) 4.40 (0.39) 9.52 (1.1) 9.00 (1.4) Active ghrelin (pg mlÀ1) 6.93 (1.81) 13.0 (2.06)* 6.81 (1.28) 13.9 (2.30)* 6.33 (1.84) 6.79 (1.35) Total ghrelin (pg mlÀ1) 310 (24.6) 396 (24.6)# 819.2 (68.5) 1037.3 (44.6)* 794.6 (96.3) 741.9 (86.9)

*Po0.05, #P ¼ 0.08 vs saline. Data are expressed as mean (s.e.m.).

International Journal of Obesity Olanzapine’s effects on meal-related ghrelin and CCK EM van der Zwaal et al 258

Figure 1 Effects of administration of olanzapine (1 mg kgÀ1) and CCK (3 mgkgÀ1) on meal size in experiment 2. Data are expressed as mean±s.e.m. *Po0.05 vs SAL/PBS, #Po0.05 vs OLA/PBS and SAL/CCK. OLA, olanzapine; SAL, saline.

middle of the light phase (experiment 1C), active and total ghrelin levels were not significantly affected (Table 2). The ratio of active to total ghrelin was not significantly different between OLA-treated rats and controls in any of the experiments (data not shown). No significant differences in leptin levels were observed between the treatment groups of each experiment (Table 2). Furthermore, subcutaneous and epididymal white adipose Figure 2 Effects of administration of olanzapine (1 mg kgÀ1) and CCK tissue weights were also comparable (data not shown). This (3 mgkgÀ1)on(a) locomotor activity and (b) body core temperature in confirmed that, concerning adiposity levels, treatment experiment 2. Data are expressed as mean±s.e.m. Arrows indicate the time of drug administration. Main effect of olanzapine treatment: *Po0.05, #P ¼ 0.05, groups were comparable within the experiments. B P ¼ 0.055. OLA, olanzapine; SAL, saline. Experiment 2: Meal size 460) ¼ 41.8, Po0.001), whereas CCK had no effect In this experiment, we investigated the effects of pretreat- (Figure 2a). Interactions were only significant between time ment with OLA or saline on the efficacy of CCK to reduce and OLA treatment (F(23, 460) ¼ 3.31, Po0.001), which was meal size at the end of the light phase. Repeated-measures explained by the finding that activity levels were signifi- ANOVA revealed a significant effect of treatment on meal cantly lower in OLA-treated rats from the first until the size (Figure 1; F(3,66) ¼ 32.7, Po0.001). Post-hoc tests fourth hour of the dark phase only (Po0.05). revealed that after saline pretreatment, administration of Repeated-measures analysis of body core temperature CCK (3 mgkgÀ1) caused a decrease in meal size compared revealed a significant main effect of time (F(23,460) ¼ with PBS injection (Po0.001). Conversely, when OLA 86.95, Po0.001) and OLA treatment (F(1,20) ¼ 8.37, pretreatment (1 mg kgÀ1) was followed by PBS injection, P ¼ 0.009) but not of CCK treatment. The interaction meal size was increased compared with saline/PBS treatment between time and OLA treatment was also significant (Po0.001). When OLA pretreatment (1 mg kgÀ1) was fol- (F(23,460) ¼ 19.62, Po0.001). Interestingly, the temperature lowed by CCK, meal size was increased compared with of OLA-treated rats was lower in the first 6 h (Pp0.05), saline/PBS treatment (P ¼ 0.014) and saline/CCK treatment whereas it was higher in the last hours of the dark phase (Po0.001), but it was significantly lower than after OLA/PBS (Po0.05, except ZT23: P ¼ 0.055). This increase in body treatment alone (P ¼ 0.006), which suggested that CCK still temperature persisted at all but one time point in the had an effect on meal size. To determine whether there was following light phase (Po0.05, Figure 2b), but disappeared any interaction between the two treatments, a two-way after the next lights-off (data not shown). repeated-measures ANOVA was performed. This revealed a significant main effect of olanzapine treatment (F(1,22) ¼ 66.6, Po0.001) and of CCK treatment (F(1,22) ¼ 19.1, Po0.001), but no significant interaction (F(1,22) ¼ 0.62, Discussion P ¼ 0.44). Thus, the effect of CCK was similar for both saline and OLA pretreatment conditions. In this study, we investigated the effect of acute administration of OLA on several meal-related parameters in rats to Experiment 2: Locomotor activity and body temperature gain insight into the effect of OLA on food intake. In rats OLA treatment had a significant main effect on locomotor treated with OLA both preprandial and postprandial ghrelin activity (F(1,20) ¼ 32.9, Po0.001), as did time (F(23, levels were increased compared with controls. Furthermore,

International Journal of Obesity Olanzapine’s effects on meal-related ghrelin and CCK EM van der Zwaal et al 259 in our behavioral paradigm, acute administration of OLA interaction between OLA and CCK treatment, which sug- caused an increase in meal size, and this effect was able to gests functional antagonism instead of a direct effect on CCK counteract the satiating effect of CCK when drugs were sensitivity. A possible explanation for this may be that coadministered. ghrelin levels in the present study were not high enough to In the rats that were about to initiate their night-time completely block the effect of CCK. In the study by Date feeding episode, acute administration of OLA caused an et al., the effect of CCK on food intake was abolished by increase in both active and total ghrelin levels (experiment pretreatment with 3 nmol (B10 mg) ghrelin intravenous in 8- 1B). Furthermore, in rats that had eaten a fixed amount of h fasted rats, and a dose of 1.5 nmol (B5 mg) was shown to food at the end of the light phase, OLA also caused a prevent a CCK-induced increase in vagal firing.22 Both these significant increase in active ghrelin levels and tended to doses are likely to have resulted in much higher ghrelin increase total ghrelin levels (experiment 1A). Conversely, levels than observed in the present study.41 Furthermore, when OLA was administered in the middle of the light phase, although the minimum effective dose of ghrelin to affect when rats hardly eat, it failed to affect ghrelin levels vagal firing was 0.3 pmol (B1 ng), the study of Date et al.22 (experiment 1C). Together, these findings indicate a selective did not report whether this lower dose was also sufficient to effect of OLA on meal-related ghrelin secretion. Measure- block the effect of CCK. Therefore, it is possible that CCK ment of postprandial levels of several gastro-intestinal sensitivity is not as clearly affected by lower levels of ghrelin, hormones in rats that had eaten a fixed amount of food although these doses of ghrelin might still affect feeding (experiment 1A) indicated that OLA does not reduce the through other pathways. secretion of any of these satiating factors. As we only Indeed, the observation that OLA did not significantly analyzed plasma levels at one time point, we cannot exclude reduce CCK sensitivity in the present study suggests that an effect of OLA occurring at an earlier or later time. the increase in meal size caused by OLA is However, both our findings are in line with a recent clinical most likely determined by pathways that are largely study in healthy volunteers in which the authors reported an independent of CCK. It is known that neuropeptide increase in total ghrelin levels at dinner time after 8 days of Y neurons in the arcuate nucleus of the hypothalamus are OLA treatment, but no reductions in the levels of CCK, PP, activated by both peripheral and central administration of PYY and GLP-1.28 ghrelin42,43 and that central administration of neuropeptide In patients, increased levels of ghrelin have also been Y is able to counteract the satiating effect of peripheral CCK reported, especially after long-term treatment with OLA.29–32 administration.44 Therefore, it is possible that the elevation Furthermore, when BMI was taken into account, ghrelin in ghrelin levels by OLA activated NPY neurons, which levels of chronically treated patients were higher than would thereby counteracted the effects of CCK. This possibility is be expected for an untreated patient with the same BMI.30,32 supported by a recent study that observed increased NPY As ghrelin levels are known to be influenced by obesity mRNA levels after acute administration of OLA.45 However, levels,33 this indicates that the effect of OLA on ghrelin levels the administered dose was very high (10 mg kgÀ1), and other is independent of weight gain. Nevertheless, clinical findings studies administering a lower dose subchronically failed to are not entirely consistent, as other authors have reported observe effects on levels of NPY mRNA levels.14,46 Therefore, decreased levels of ghrelin,34–38 especially after short-term it remains to be determined whether OLA affects NPY levels treatment with OLA. at the dose used in the present study. Little is known about the regulation of peripheral ghrelin Finally, it should be noted that, in addition to secretion, and the mechanisms underlying the effect of OLA ghrelin and CCK, many other factors have a role in meal on peri-prandial ghrelin levels, therefore, remain unclear. termination and may therefore be involved in the effect However, increased ghrelin levels were also reported in rats of OLA on food intake, including the response to receiving an injection of clozapine, an antipsychotic closely hormones such as leptin, insulin, GLP-1 and PYY.11,12 related to OLA, and after administration of an alpha- Additional experiments are necessary to determine the adrenergic receptor antagonist.39 This suggests that the effect of OLA on the sensitivity to other hormones and effects of clozapine and OLA may be due to their antag- peptides. onistic effect at adrenergic receptors.40 In addition to the effect on meal size, acute administration The effect of acute administration of OLA on meal size in of OLA also caused a (temporary) reduction in locomotor the behavioral paradigm used in this study replicates our activity and body core temperature, which is similar to findings with continuous administration of OLA, which previous observations.16,47–50 Interestingly, the decrease in confirms that the paradigms are comparable.16 Moreover, it body temperature was followed by a much longer-lasting is in line with the findings of other authors.13–15 The increase increase in temperature, which continued into the following in peri-prandial ghrelin levels by OLA may partially underlie light phase. Reactive hyperthermia was previously also this effect. As mentioned earlier, in the study by Date et al.,22 reported after OLA was discontinued in rats that had been ghrelin pretreatment was able to completely abolish the treated continuously for 18 days.51 Our data indicate that effect of CCK, indicating a reduction in CCK sensitivity such a compensatory response can already occur after a by ghrelin. However, we failed to observe a significant single injection of OLA.

International Journal of Obesity Olanzapine’s effects on meal-related ghrelin and CCK EM van der Zwaal et al 260 Taken together, this study used a paradigm of acute OLA 13 Cooper GD, Goudie AJ, Halford JC. Acute effects of olanzapine administration in rats to investigate the mechanisms on behavioural expression including the behavioural satiety underlying the effects of OLA on food intake. It shows sequence in female rats. J Psychopharmacol 2010; 24: 1069–1078. 14 Davoodi N, Kalinichev M, Korneev SA, Clifton PG. Hyperphagia that administration of this drug leads to an increase in and increased meal size are responsible for weight gain in rats peri-prandial ghrelin levels and this effect may partially treated sub-chronically with olanzapine. Psychopharmacology underlie the finding that OLA counteracts the satiating effect (Berl) 2009; 203: 693–702. of CCK. 15 Thornton-Jones Z, Neill JC, Reynolds GP. 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