Student Jacolien C. Graver (1726064)

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Olanzapine in roman rats

Effects of olanzapine on body weight, adiposity and insulin sensitivity in female Wistar, Roman Low Avoidance and Roman High Avoidance rats.

Student

Jacolien C. Graver (1726064)

Supervisor

Drs. Simon S. Evers

Prof. Anton J.W. Scheurink

Departement of Neuroendocrinology, University of Groningen Subject

Master report Biomedical Sciences, University of Groningen Date

June 29, 2012

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Abstract

Introduction: Olanzapine is an atypical antipsychotic drug used for treatment of schizophrenic patients.

A major side effect of olanzapine is the substantial weight gain. Olanzapine acts predominantly antagonistic on serotonin and dopamine receptors, but also on histamine, muscarinic and α-adrenergic receptors. These multiple action sites make it difficult to elucidate the mechanisms of action of olanzapine. To our knowledge a valid animal model for schizophrenia has not been identified. Therefore this study looked of the effects of olanzapine on body weight, adiposity and insulin sensitivity in Wistar, passive Roman low- (RLA) and proactive Roman high avoidance (RHA) rat. Also effects of OLZ treatment in an active versus a sedentary environment were tested in RLA and RHA rat.

Methods: Olanzapine was administered twice daily (2 mg/kg) at the beginning and in the middle of the dark phase, resulting in 4 ml/kg olanzapine per animal per day. On daily basis body weight, food intake and water intake were measured and an intravenous glucose tolerance test was performed to measure insulin and glucose response. Furthermore post mortem analysis was performed. The first project was performed with female Wistar rats. The second and third projects were performed with RLA and RHA rats. Running wheel activity was measured during the second project, whereas cage activity and body temperature were measured in the third project.

Results: The first project showed significant increased body weight and insulin levels following olanzapine treatment. During the second project olanzapine treatment of RHA rats showed significantly increased body weight, total fat and visceral fat. Furthermore, running wheel activity was significantly decreased. In RLA rats OLZ only significantly lowered running wheel activity. In the third project cage activity and body temperature were significantly decreased by OLZ treatment in both RLA and RHA rats.

Conclusion: Most effects of olanzapine were seen in RHA rats in an active environment, with increased body weight and adiposity. Next to weight gain, only Wistar rats displayed decreased insulin sensitivity.

The olanzapine induced decrease of activity was independent of coping style and environment. Also the hypothermic effect of olanzapine was independent of coping style. However, the used animal models in this study did not display all the side effects seen in schizophrenic patients. Therefore, future research should aim at searching a valid animal model which at least displays the side effects of olanzapine on body weight, adiposity and insulin sensitivity.

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List of abbreviations

ACTH = adrenocorticotrophic hormone APD = antipsychotic drug

BAT = brown adipose tissue b.i.d. = bis in die, twice daily BT = body temperature BW = body weight FI = food intake

HPA axis = hypothalamus-pituitary-adrenal axis LBM = lean body mass

OLZ = Olanzapine

RLA = roman low avoidance RHA = roman high avoidance s.c. = subcutaneous

UCP1 = uncoupling protein 1 WI = water intake

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1 Introduction

Olanzapine (OLZ) is a widely prescribed antipsychotic drug (APD) used for treatment of schizophrenia (Cooper et al., 2005). It reduces symptoms like hallucinations, delusions and psychotic episodes. Before the introduction of atypical APD, first generation APDs were used. These drugs induced, through the blockage of dopamine D2 receptor, extrapyramidal side effects with among others parkinsonian symptoms (Reynolds et al., 2010). OLZ is part of second generation APDs that have enhanced efficacy and reduced adverse effects compared to first generation APD’s. However, these atypical APDs have a major side effect, they induce substantial weight gain (Cooper et al., 2007; Houseknecht et al., 2007;

Caballero et al., 2003). This weight gain leads to metabolic deregulations including insulin resistance, hyperglycemia, dyslipidemia and can rapidly result in obesity which increases the risk of developing type II diabetes (Reynolds et al., 2010; Houseknecht et al., 2007; Albaugh et al., 2006). Furthermore, weight gain and obesity increase the risk of hypertension, respiratory problems and cardiovascular disease.

Schizophrenic patients already have a higher prevalence of type II diabetes, respiratory complications, cardiovascular disease and a decreased life expectancy compared to the general population (Fell et al., 2005; Houseknecht et al., 2007; Rummel-Kluge et al., 2010). These risk factors are likely to exacerbate in schizophrenic patient with APD treatment.

1.1 OLZ - side effects

In humans, some of the observed weight gain may be caused by the sedative effects of OLZ. However OLZ induced weight gain is also associated with enhanced appetite and increased food intake. Thus, the observed weight gain and enhance adiposity might be caused by increased food intake and/or reduced physical activity (Cooper et al., 2005). However, the underlying mechanisms of weight gain and metabolic deregulation are still unclear (Evers et al., 2010, Reynolds et al., 2010). Furthermore, previous studies of OLZ treatment in rats showed acute hypothermia (Evers et al., 2010; Stefanidis et al.,2009;

Goudie et al., 2007). This hypothermic effect could be the result of the direct inhibitory effect of OLZ on sympathetic action and brown adipose tissue (BAT) thermogenesis. A necessary counterpart of BAT thermogenesis is uncoupling protein 1 (UCP1). This protein plays a role in uncoupling mitochondrial respiration from ATP synthesis. OLZ decreases expression of UCP1 in BAT, thereby indirectly impairing BAT thermogenesis. Furthermore, it has been shown that OLZ blocks orexin-A induced hyperthermia (Monda et al., 2008). These data from previous studies imply a centrally regulated origin for OLZ induced hypothermia, directly related to the orexin-A system at the lateral hypothalamus.

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1.2 OLZ – Pharmacology

It is known that OLZ acts predominantly antagonistic on 5-HT2A/C and dopamine receptors, but also on histamine, muscarinic and α-adrenergic receptors. These receptors are distributed throughout the CNS neuronal networks, digestive tract, skeletal muscle and adipose tissue. Because of this widespread distribution of receptors throughout the body, OLZ may have multiple central and peripheral action sites (Evers et al., 2010, Reynolds et al., 2010). This makes it difficult to elucidate the mechanisms of action of OLZ.

1.3 OLZ - Gender differences

Unlike the human situation, OLZ induced weight gain in rats has gender-dependent effects. Compared to males, female rats display a more robust weight gain following OLZ treatment. However, male rats are not completely unaffected by OLZ treatment. They do show metabolic effects in the absence of body weight gain (Albaugh et al., 2011; Cooper et al., 2007; Davey et al., 2012). One example is the enhanced adiposity seen in male and female rats as well as in other species like dogs and humans (Ader et al., 2005; Graham et al., 2005). The cause of these gender differences is still unknown, but it shows the importance of choosing valid animal models for OLZ research. Most research on OLZ treatment has been done in male rats although female rats seem to mimic the clinical situation better. For this reason, our study used female rats.

1.4 Animal model

Furthermore, in humans susceptibility for metabolic disorders differs largely among individuals. Most animal studies ignore this important individual variation (Boersma et al., 2011a). As mentioned before, schizophrenic patients are more susceptible to develop type II diabetes. Most animal models that are currently used do not have this innate tendency to develop type II diabetes. Therefore translation to the human situation loses validity. Boersma et al. showed that the effect of drug treatment can be dependent on coping style. In this study coping style was defined as: “a coherent set of behavioral and physiological stress responses that are consistent over time and that characterize a certain group of individuals” (Boersma et al., 2011a; Boersma et al., 2011b). To enhance the validity of our research, Roman Low and High Avoidance rats (RLA and RHA, respectively) were used. This animal model seems to represent some of the schizophrenic characteristics better than standard animal models.

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1.5 Roman avoidance rats

Boersma et al. showed that a RLA rat is metabolically different from a RHA rat and may serve as a non- obese animal model for insulin resistance (Boersma et al., 2009). Roman Avoidance rats were originally selected and bred from a Wistar stock on their performance in a shuttle box with a two-way active avoidance test (Bignami, 1965). While RLA rats showed poor acquisition and passive behavior in this test, RHA rats showed rapid acquisition and active behavior. Indeed, research showed that RLA rats have a passive coping style with increased parasympathetic and reduced sympathetic reactivity. They show behavioral flexibility, an increased stress response, freezing behavior, anxious behavior and increased emotional responsiveness. Furthermore RLA rats have an increased sensitivity of the hypothalamus- pituitary-adrenal (HPA) axis. This leads to increased corticosterone, corticotrophin and adrenocorticotrophic hormone (ACTH) secretion. It was also found that RLA rats are more prone to develop metabolic diseases like insulin resistance (Boersma et al., 2011a, b; Boersma et al. 2009;

Steimer et al., 2005). In contrast to RLA rats, RHA rats have an active coping style with increased sympathetic reactivity and increased dopamine receptor expression. They are characterized by low emotional reactivity, impulsive behavior and little anxiety. They show compulsive behavior and have higher levels of novelty seeking in a new environment. Other personality traits of the RHA rats are that they show low HPA-axis reactivity and a greater preference for rewarding substances (Boersma et al., 2011a; Boersma et al., 2009; Steimer et al., 2005). Furthermore, under baseline conditions RLA rats have high insulin levels while RHA rats have relative low insulin levels (Boersma et al., 2011a). It will be interesting to see how insulin levels change when animals, with free access to a running wheel, are treated with OLZ.

1.6 Research aim

The aim of the current study is to investigate how OLZ treatment affects body weight (BW), adiposity and insulin sensitivity in RLA and RHA rats. Beside these parameters we also looked at effects of OLZ treatment on daily food and water intake, activity, temperature and glucose and insulin levels.

Since schizophrenic patients have a higher prevalence of developing insulin resistance and RLA rats have higher susceptibility of developing insulin resistance we use the RLA rat as an animal model to test the metabolic effects of OLZ treatment. Therefore, our first hypothesis is that RLA OLZ rats gain more weight, have increased adiposity and decreased insulin sensitivity compared to RLA control rats.

Although RHA rats do not have an increased susceptibility of developing insulin resistance, RHA rats do have increased dopamine expression and a great preference for rewarding substances. These

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8 characteristics are also seen in schizophrenic patients. Therefore we expect OLZ treatment in RHA rats

to show the same side effects seen in schizophrenic patients. That is why our second hypothesis is that RHA OLZ rats gain more weight, show increased adiposity and decreased insulin sensitivity compared to RHA control rats.

Research showed that when RLA and RHA rats were placed in a cage with free access to a running wheel, RLA rats showed higher activity levels compared to RHA rats. This increased activity led to normalization of the plasma insulin response in RLA rats. Furthermore, RHA rats seemed to be less sensitive to external cues. Therefore Boersma et al. 2011a speculated about RLA rats being more susceptible to life style intervention programs than RHA rats. Our third hypothesis is that the response of BW, adiposity and insulin sensitivity to OLZ will be the same in RHA rats regardless of the environment. At last the fourth hypothesis, RLA rats are more capable of diminishing the effects of OLZ on BW, adiposity and insulin sensitivity in a sedentary environment.

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2 Materials & Methods

2.1 Animals

Animals in all three projects had free access to standard lab chow (3.8 kcal/g, Hope Farms, RMH-B knaagdier korrel, Arie Blok Diervoeding, Woerden, NL) and water. Room temperature and humidity were controlled (T= 20±2 ˚C, humidity 60%) with a 12-12 h light-dark cycle (lights on at 23:00h). All animals were habituated to the housing environment one week before undergoing surgery. Baseline measurement started at least one week after surgery when animals reached their presurgical body weight. One day before drug treatment all groups were given medium fat diet with lard (4.7 kcal/g; 45%

fat, Arie Blok Diervoeding, Woerden, NL) to stimulate weight gain. All animal experimental procedures were approved by the Animal Experimentation Committee of the University of Groningen.

In the first project twelve adult female Wistar rats (Harlan, Horst, NL), with an average weight of 221 ± 2.8 g at the beginning of the experiment, were housed individually in clear plexiglass cages (25x25x30cm). The cages were filled with wood chipped bedding on a plastic floor.

During the second project twelve female RLA rats (257 ± 3.2 g; at start of the study) and twelve female RHA rats (255 ±3.8 g; at start of the study) were originally obtained from Geneva, Switzerland and bred locally at the animal care facility of the university of Groningen, the Netherlands. The RLA rats and the RHA rats were both split into a control and drug treated group (n=6/group). All animals were housed individually in Nalgene polycarbonate running wheel cages (50x27x36cm) filled with wood chipped bedding and they had free access to a running wheel (diameter 27cm, mini mitter, Oregon, USA).

The third project also used twelve female RLA rats (279 ± 5.4 g; at start of the study) and twelve female RHA rats (262 ± 7.0 g; at start of the study). Both RLA and RHA rats were split into a control and drug treated group (n=6/group). All animals were housed individually in clear Plexiglas cages (25x25x30cm) filled with wood chipped bedding on a plastic floor.

2.2 Surgery

All the animals underwent surgery to place a gastric catheter and two indwelling jugular vein catheters.

This is respectively for stress-free intra gastric drug administration and blood sampling in freely moving animals. During surgery animals were sedated using Isoflurane-O2/N2O gas.

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10 The gastric catheter (1.40 mm OD, 0.80 mm ID) was inserted at the level of the corpus through the

gastric wall, extending 0.5 cm into the gastric lumen. The catheter was pulled subcutaneously towards the head where it was connected to a metal bow. This metal bow was fixed to the skull with dental cement and screws (Wielinga et al., 2005). The jugular vein catheters (0.95 mm OD, 0.50 mm ID and 0.64 mm OD, 0.28 ID) were inserted into the right and left jugular vein and kept in place with a ligature. Both catheters were pulled subcutaneously towards the head where they were connected to metal bows. The metal bows were fixed to the skull with dental cement and screws. To prevent the formation of blot cloths, catheters were filled with a polyvinyl pyrrolidone-heparin solution (Steffens, 1969). For analgesia animals were given 0.1 ml Finadine subcutaneously (s.c.). To prevent infection the animals got s.c. 0.25 ml penicillin. Next to the two jugular vein - and gastric catheters, animals of project 3 also got a transmitter (model TA10TA-F40, Data Sciences, St. Pail, MN) in the abdominal cavity. This transmitter continuously registrated body temperature and activity (Meerlo et al., 1996) in freely moving animals.

2.3 Experimental design

The study consisted of three projects. For all three projects baseline measurements started 7 days prior to drug administration. During baseline period animals got intragastric saline injections twice daily. On day -1 animals were switched from standard lab chow to medium fat food. From day 0 the drug treated animals got injections of 2 ml/kg olanzapine bis in die (b.i.d.) while control animals continued to get saline injections b.i.d. During all three projects body weight, food intake and water intake were measured prior to the dark phase (11.00h). Next to these standard measurements running wheel activity per minute was measured during the second project. In the third project core body temperature and cage activity data (sedentary) were collected every 5 minutes. In all three projects an intravenous glucose tolerance test (IVGTT) was performed at day 14. The first project continued measurements for 7 days after this IVGTT. Post mortem analysis was then performed on day 21. During the second and third project animals were sacrificed after the IVGTT (day 14) to perform post mortem analysis.

2.4 Drug

During this study the drug Olanzapine (OLZ) was used. The drug, as powder, was kindly provided by Abott (Fournier Laboratory, France). OLZ was dissolved in saline (0,9% NaCl) using 1M HCl. To reach a pH of 7, 1M NaOH was added to the OLZ solution. Saline was added to the solution to obtain a concentration of 1 mg/ml Olanzapine. Each administration contained 2 mg/ml Olanzapine. Saline or OLZ was given b.i.d. prior to the dark phase and in the middle of the dark phase. This resulted in daily administration of 4 ml/kg OLZ per animal.

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2.5 Circadian registration

From the animals of project 3, core body temperature (◦C) and cage activity (arbitrary unit) were recorded during the entire experiment. This was done using radio telemetry in which telemetry signals were received by separate plates (model RA1010, Data Sciences) underneath each cage. Every 5 min data were collected and analyzed using specialized recording and analyses software (Dataquest IV, Data Sciences) (Meerlo et al., 1996).

2.6 Intravenous glucose tolerance test

An intravenous glucose tolerance test (IVGTT) was performed to measure the insulin response in each individual animal. During an IVGTT glucose is given intravenously, to test how the body responds by releasing insulin into the blood. In order to measure the amount of insulin blood samples were collected at different time points.

Before the actual onset of the test, rats could get accustomed to the infusion and blood sampling procedure (Steffens, 1969). Three hours before the start of the IVGTT food was removed. One hour before the start of the test baseline blood samples were collected from the catheter and placed into plastic tubes which were stored on ice (time point -60). During the IVGTT glucose infusion of 15 mg/min was given in 3 mL saline solution. This is a physiological dose that mimics the glucose response after a large meal (Strubbe and Bouman, 1987). Glucose infusion started at time point 0 and continued for 30 min. Immediately after the start of glucose infusion blood samples of approximately 2 mL each were obtained at time points 0, 5, 10, 15, 20, 30, 45, 60 and 120 min. After each blood sample, approximately 1 ml of heparin was used to prevent the formation of blood clots and physiological saline was used to flush the catheter. All blood samples were placed into plastic tubes with 10 µL EDTA (0.09 g/mL) to avoid blood clotting and were stored on ice. For glucose analysis, 50 µL of the full blood samples were stored with 450 µL heparin solution (2%) at -20 ˚C. Blood glucose levels were determined using the ferry- cyanide method (Hoffman et al., 1937) in a Technicon auto analyzer. The remaining blood samples were centrifuged for 15 min at 1880Xg. Plasma was stored at -20 ˚C for insulin determination. Plasma insulin levels were measured using commercial radioimmunoassay (RIA) kits (Linco Reasearch) (Boersma et al., 2011c).

2.7 Post mortem analysis

In the first project animals were sacrificed 7 days after the IVGTT (day 21). During the second and third project, animals were sacrificed two hours after the last blood samples of the IVGTT (day 14). Animals were put under anesthesia (isoflurane-O2/N2O gas) and sacrificed by decapitation. Trunk blood (± 10 mL)

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12 was collected in plastic tubes filled with 100 µL EDTA and centrifuged for 15 min at 2500 rpm. Following

decapitation brains were excised and frozen with liquid nitrogen and stored at -80˚C for further analysis.

Peritoneal fat, retroperitoneal fat, gastro-intestinal fat and brown adipose tissue were carefully excised and weighed. Liver, kidneys and adrenals were taken out and weighed. The skin with subcutaneous fat was separated from the carcass. Carcass, skin and liver were dried in an oven at 80˚C for 5 days. After drying, the tissues were put in a petroleum based Soxlet fat extractor to dissolve the remaining fat and dried for another day. The dried tissues were weighed before and after fat extraction in order to measure the amount of fat content of each tissue.

2.8 Data analysis

All data are expressed as averages ± standard error of the mean. Activity data were analyzed as a change in percentage of activity relative to baseline per individual. Baseline was calculated as the average activity per day from day -7 until day -1 per individual. This average was set as 100%. Activity data were shown as percentage of this baseline value. Body composition parameters were analyzed as a percentage of total BW measured on the day of post mortem analysis.

Statistical analysis was performed using repeated measures (rm) analysis of variance (ANOVA) between subjects for time dependent analyses. To calculate significance at different time points of treatment One-way ANOVA was used. Where appropriate, paired samples t-test was performed on each group. All statistical analyses were performed in SPSS 18.0.3., outcomes were regarded significant with a significance level of 5 % or lower.

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3 Results

3.1 Project 1: Olanzapine in Wistar rats

3.1.1 Body weight

Figure 1 displays the change in body weight (BW) of female wistar rats during baseline and experimental period. On day 0, the start of the experimental period, BW of control animals was 242 ± 3.52g and 236 ± 5.92g for OLZ treated rats. Although both groups gain weight during the experimental period, the OLZ treated group seemed to increase their BW more rapidly.

Statistical analysis showed that OLZ treatment (4mg/kg) caused an increase in BW, with significant effect on day 10 until day 13 (one-way ANOVA, P<0.05). Day 14 just failed to reach significance (one-way ANOVA, P=0.059). From day 14 BW of OLZ treated animals seemed to reach a plateau while the control group kept increasing BW slightly.

3.1.2 Food intake

During baseline period there were no differences in food intake (FI) between the groups (fig. 2). Both groups display a decrease in the beginning of the baseline period after which they stabilize. From day -1 all the animals increase their FI on the medium fat diet. In both groups FI gradually decreased during the course of the experimental period. In the first two weeks of the experimental period the OLZ group

-15 0 15 30 45

-7 0 7 14 21

Δ BW (g)

Time (days)

∆ Body Weight

Control

OLZ * *

*

Figure 1. Delta body weight gain of control and Olanzapine treated female Wistar rats. OLZ= olanzapine.

*Significant difference versus control group (P<0.05). Data shown represent mean ± s.e.m.

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14 seems to have a higher caloric intake than the control group. However, in the last week this effect

disappeared. OLZ treatment showed significant increases in daily caloric intake for day 5, 6, 8 and 9 (one-way ANOVA, P<0.05). Caloric intake on day 4 just failed to reach significance (one-way ANOVA, P=0.061).

Figure 3 displays the cumulative FI of the experimental groups. In week 1 FI was significantly higher for control and OLZ groups compared to their baseline period (paired samples t-test, P<0.01). In week 2 both groups reduced their FI to comparable baseline levels. In addition, during week 3 food intake of both control and OLZ animals even further decreased. Looking at the effect of OLZ treatment cumulative FI was higher compared to control in week 1 and 2.

0 50 100

-7 0 7 14 21

FI (kcal)

Time (days)

Daily Caloric Intake

Control OLZ

*

* *

*

Figure 2. Daily food intake (kcal) of control and Olanzapine treated female Wistar rats. OLZ= olanzapine.

*Significant difference versus control group(P<0.05). Data shown represent mean ± s.e.m.

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15 3.1.2 Water intake

OLZ treatment had no effect on daily water intake (WI) (fig 4). Looking at the graph, both groups had a higher WI during baseline period compared to the experimental period. On day -1 there is a sudden decrease in WI for both groups.

0 100 200 300 400 500 600

baseline Week 1 Week 2 Week 3

Food intake (kcal)

Cumulative food intake

Control OLZ

◊

0 20 40

-7 0 7 14 21

WI (ml)

Time (days)

Daily Water Intake

Control OLZ

Figure 3. Cumulative food intake (kcal) of control and Olanzapine treated female Wistar rats. Results are separated into 4 weeks. OLZ= olanzapine. ◊ Significant difference week 1 versus baseline (P<0.01). Data shown represent mean ± s.e.m.

Figure 4. Daily water intake (ml) of control and Olanzapine treated female Wistar rats. OLZ= olanzapine.

Data shown represent mean ± s.e.m.

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16 3.1.3 Glucose and insulin

Glucose and insulin responses to an IVGTT in Wistar rats are shown in figure 5A and B. Glucose responses showed no difference between the OLZ and control group. At baseline, blood glucose levels started approximately at 4.24 ± 0.47 mM for control group and 3.48 ± 0.41 mM for OLZ groups. During infusion of glucose the blood glucose levels increased to a maximum of 12.60 ± 1.07 mM for the control and 10.59 ± 1.10 mM for the OLZ group. After termination of the infusion, glucose levels returned towards baseline within 30 min.

Insulin data from the IVGTT showed a significant increased insulin levels for the OLZ group (repeated measures, F1,12=7.992, P<0.05). Blood insulin levels of control group were 0.93 ± 0.16 ng/ml while blood insulin of OLZ group was 1.70 ± 0.32 ng/ml at baseline. During the 30 minutes of glucose infusion, insulin in the OLZ group was significantly increased (one-way ANOVA, P<0.05).While control group increased insulin to a maximum of 9.99 ± 2.25 ng/ml, OLZ treated animals increased insulin to a maximum of 22.85

± 2.17 ng/ml insulin.

0 4 8 12

-60 0 60 120

Glucose (mM)

Time (min)

Glucose Response

Control OLZ A

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17 3.1.4 Body composition

OLZ treatment had no significant effect on fat distribution of peritoneal and retroperitoneal fat (fig 6).

However, OLZ treated animals did show a tendency towards having higher percentages fat.

0 5 10 15 20 25 30

-60 -30 0 30 60 90 120

Insulin (ng/ml)

Time (min)

Insulin

Control OLZ

*

* B

0 2 4

Peri Retro Peri+Retro

% Fat of body weight

Adiposity

Control OLZ

Figure 5. Glucose and insulin response to an IVGTT in control and olanzapine treated female Wistar rats.

A: Glucose response. B: Insulin response. OLZ= olanzapine, IVGTT= intravenous glucose tolerance test.

Figure 6. Adiposity of control and Olanzapine treated female Wistar rats. Adiposity is shown as % fat of total body weight. OLZ= olanzapine. Data shown represent mean ± s.e.m.

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3.2 Project 2: Olanzapine in RLA and RHA rats in an active environment

Figure 7A and B display the change in BW from day 0, in respectively RLA rats and RHA rats under control conditions and with OLZ treatment in cages with a running wheel. At the start of the experimental period BW of RLA control animals was 276.3 ± 4.5 g and BW of RLA OLZ animals was 264.7

± 9.1 g. For RHA control animals BW at day 0 was 264.8 ± 6.7 g and BW of RHA OLZ animals was 253.3 ± 2.9 g. Although the RLA control en OLZ groups differ in part of the baseline measurements, they follow the same pattern during the entire experimental period. The groups showed an evident increase in BW from day 0 until day 5 after which they stabilized their BW.

Looking at changes in BW of RHA control and OLZ groups (fig. 7B) there were no differences in baseline period. Following drug administration repeated measures showed that the OLZ treated group was significantly heavier than the control group (repeated measures, F1,10= 27.909, P<0.001). Both groups showed the same increase from day 0 until day 3. After this increase both groups started to differ. This difference was significant on day 5 until day 12 (one-way ANOVA, P<0.01). The control group seemed to stabilize BW but showed an increase in the end.

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19 -25

-20 -15 -10 -5 0 5 10 15 20 25 30

-7 0 7

Gain (g)

Time (days)

∆ Body weight RLA

RLA Control RLA OLZ A

-25 -20 -15 -10 -5 0 5 10 15 20 25 30

-7 0 7

Gain (g)

Time (days)

∆ Body weight RHA

RHA Control RHA OLZ

* * * * * * * B

Figure 7. Delta body weight gain of female RLA and female RHA rats. A: delta body weight gain of control and olanzapine treated RLA rats. B: delta body weight gain of control and olanzapine treated RHA rats.

OLZ= olanzapine. *Significant difference versus control group (P<0.01). Data shown represent mean ± s.e.m.

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20 3.2.2 Food intake

Figure 8A and B showed the daily FI of RLA and RHA rats under control conditions and with OLZ treatment. During baseline conditions both RLA and RHA rats showed no difference in FI between control and OLZ animals. At day -1 RLA and RHA rats rapidly increased their FI on a medium fat diet, which decreased again at day 0. During the experimental period control and OLZ treated RLA animals showed a gradual decline in FI. Furthermore, for the RLA rats there were no major differences in daily FI between the two groups. During the experimental period, food intake of OLZ treated RHA animals was higher from day 4 until day 7, with significant effect on day 5 (one-way ANOVA, P<0.01). Furthermore the OLZ treated animals showed a decrease in their FI at the end of the treatment, which was significantly lower than controls on day 12 and 13 (one-way ANOVA, P<0.05).

Comparing RLA control with RHA control animals, RLA control rats ate significantly more (repeated measures, F1,10=7.748, P<0.05). This difference disappeared when OLZ was administered.

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21 0

40 80 120 160

-7 0 7

Food Intake (kcal)

Time (days)

Daily Food Intake RLA

0 40 80 120 160

-7 0 7

Food Intake (kcal)

Time (days)

Daily Food Intake RHA

RHA Control RHA OLZ B

**

*

Figure 8. Daily food intake (kcal) of female RLA and female RHA rats. A: food intake of control and olanzapine treated RLA rats. B: food intake of control and olanzapine treated RHA rats. OLZ= olanzapine. *Significant difference versus control group (P<0.05). **Significant effect versus control group (P<0.01). Data shown represent mean ± s.e.m.

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22 There were no effects of OLZ treatment in cumulative FI of RLA and RHA rats during the project (fig 9).

Comparing week 1 with baseline showed that all groups ate significantly more during week 1 (paired- samples t-test, P<0.01). However, this difference disappeared during week 2, where all groups decreased their FI to comparable levels. Cumulative FI of RLA control en RHA control was only different in week 1, with RLA control eating significantly more than RHA control (one-way ANOVA, P<0.01).

Treatment with OLZ diminished this effect between RLA and RHA animals, with no statistical differences between these groups.

3.2.3 Water intake

WI of RLA rats was significantly higher than WI of RHA rats (one-way ANOVA, P< 0.01) (fig. 10). There were no differences in WI between control and OLZ treated animals for both RLA and RHA. Week 1 showed a decrease in WI compared to baseline for all groups which persisted in week 2. This decrease in WI reached significance for RHA control animals in both week 1 and 2 (paired samples t-test, P< 0.01).

0 300 600 900

Baseline Week 1 Week 2

Food (kcal)

Cumulative Food Intake

RLA Control RLA OLZ RHA Control RHA OLZ

◊

†

Figure 9. Cumulative food intake (kcal) of control and olanzapine treated female RLA and control and olanzapine treated female RHA rats. Results are separated into 3 weeks. OLZ= olanzapine. ◊Significant difference of week 1 versus baseline (P<0.01). †Significant difference of RLA control versus RHA control (P<0.01). Data shown represent mean ± s.e.m.

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23 3.2.4 Glucose

Glucose responses to an IVGTT in RLA and RHA rats are shown in figure 11A and B. Glucose responses of RLA rats showed no difference between OLZ treated animals and controls. Baseline blood glucose levels started at 5.63 ± 0.28 mM RLA control and 5.55 ± 0.25 mM for RLA OLZ animals. During glucose infusion blood glucose levels increased to a maximum of 10.13 ± 0.74 mM for RLA control and 10.30 ± 0.99 mM for RLA OLZ treated animals.

The glucose response of RHA OLZ rats seemed to be higher than RHA control rats, but this difference did not reach significance. Furthermore, calculating the area under the curve (AUC) neither showed significance (see Appendix fig. 1). RHA controls started with blood glucose levels of 6.20 ± 0.21 mM and RHA OLZ animals had basal blood glucose levels of 5.30 ± 0.15 mM. During glucose infusion RHA control animals increased their blood glucose levels to a maximum of 9.13 ± 1.00 mM, whereas RHA OLZ animals had maximum glucose levels of 11.99 ± 0.69 mM. After termination of the infusion, glucose levels of all groups turned towards baseline within 30 minutes.

Comparing RLA and RHA animals, there were no differences in glucose levels of both control and OLZ groups.

0 100 200 300 400

Baseline Week 1 Week 2

Water (mL)

Cumulative Water Intake

† †

† † †

◊ ◊

†

Figure 10. Cumulative water intake of control and olanzapine treated female RLA and control and olanzapine treated female RHA rats. Results are separated into 3 weeks. OLZ= olanzapine. †Significant difference of RLA groups versus RHA groups (P<0.01). ◊ Significant difference of RHA control versus baseline (P<0.01). Data shown represent mean ± s.e.m.

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24 3.2.5 Insulin

There were no differences in insulin response between control and OLZ treated animals for both RLA and RHA rats (fig. 12). Plasma insulin levels started at 0.75 ± 0.21 mM for RLA control and 0.97 ± 0.15 mM for RLA OLZ. Insulin levels increased to a maximum of 7.81 ± 1.01 ng/ml for RLA control and 7.39 ± 0.93 ng/ml for RLA OLZ.

RHA control animals started with plasma insulin levels of 0.76 ± 0.12 mM and RHA OLZ had baseline insulin of 0.41 ± 0.16 mM. During glucose infusion insulin increased to a maximum of 6.86 ± 0.82 ng/ml for RHA controls and 7.23 ± 0.62 ng/ml for RHA OLZ rats. Furthermore, RLA OLZ animals had significant

4 8 12

-60 0 60 120

Glucose (mM)

Time (min)

Glucose Response RLA

4 8 12

-60 0 60 120

Glucose (mM)

Time (min)

Glucose Response RHA

Figure 11. Glucose response to an IVGTT in RLA and RHA rats. A: Glucose response of control and olanzapine treated RLA rats. B: Glucose response of control and olanzapine treated RHA rats. OLZ= olanzapine. Data shown represent mean ± s.e.m.

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25 higher baseline insulin levels compared to RHA OLZ animals (one-way ANOVA, P< 0.05). This difference

was not present in control animals.

3.2.6 Activity

Running wheel activity was measured during project 2 and is shown in figure 13. During baseline period RLA control rats were more active than RHA control rats (one-way ANOVA, P<0.01) but during experimental period this difference failed to reach significance (one-way ANOVA, F1,11= 4.201, P=0.068).

Activity levels during baseline between RLA control and OLZ and between RHA control and OLZ did not 0

1 2 3 4 5 6 7 8 9 10

-60 -30 0 30 60 90 120

Insulin ng/ml

Time (min)

Insulin response RLA

0 1 2 3 4 5 6 7 8 9 10

-60 -30 0 30 60 90 120

Insulin ng/ml

Time (min)

Insulin response RHA

Figure 12. Insulin response to an IVGTT in RLA and RHA rats. A: Insulin response of control and olanzapine treated RLA rats. B: Insulin response of control and olanzapine treated RHA rats. OLZ= olanzapine. Data shown represent mean ± s.e.m.

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26 differ. Treatment with OLZ abolished the existing difference between RLA and RHA. In addition, during

the experimental period, both RLA OLZ and RHA OLZ showed diminished running wheel activity as compared to their controls (one-way ANOVA, P<0.01; one-way ANOVA, F1,11= 4.436, P=0.061 respectively). Analysis with Paired-Samples T-test showed that OLZ groups of both RLA and RHA significantly decreased their running wheel activity compared to their baseline period (paired samples t- test, P<0.05). Both control groups increased their activity compared to baseline period. Remarkably, while variance in both control groups increased during experimental period, variance in OLZ groups decreased.

Running wheel activity is shown in figure 14A for RLA and figure 14B for RHA. To see how animals changed activity during drug treatment, activity is shown as percentage of baseline for each group.

Running wheel activity of RLA control and OLZ animals was comparable in baseline period. During drug treatment these groups differed significantly (repeated measures, F1,10= 12.326, P<0.01). ANOVA analysis showed significant decreased activity for RLA OLZ animals on day 1 and day 7-13 (one-way ANOVA, P<0.05).

During the entire experimental period the RHA OLZ group showed a significant lower level of running wheel activity compared to the RHA control group (repeated measures, F1,10= 30.129 P<0.001).

0 2000 4000 6000 8000 10000

baseline experimental

revolutions

Running wheel activity

RLA control RLA OLZ RHA control RHA OLZ

†

*

◊

#

Figure 13. Running wheel activity (revolutions) of control and olanzapine treated RLA and control and olanzapine treated RHA rats. Results are separated on baseline and experimental period. OLZ= olanzapine. *Significant difference versus control group (P<0.05). † Significant difference of RLA control versus RHA control group (P<0.01). ◊ Significant difference of RLA OLZ experimental versus baseline (P<0.05). # Significant difference RHA OLZ experimental versus baseline (P<0.05). Data shown represent mean ± s.e.m.

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27 Statistical analysis showed significant lower activity levels for RLA OLZ animals compared to their

controls for all experimental days except for day 1 where P=0.053 (one-way ANOVA, day 0, 2, 3 P<0.05;

one-way ANOVA, day 4-13 P<0.01). Furthermore, during the experimental period variance within the OLZ group of both RLA and RHA decreased, whereas control animals showed more variance.

Figure 15 displays the running wheel activity of the RLA and RHA groups separated for dark and light phase. All groups were more active during the dark phase. In both OLZ treated groups running wheel activity during dark phase was significantly lowered compared to their corresponding controls (one-way

0 50 100 150 200 250

-7 0 7

% of baseline

Time (days)

Running wheel activity RLA

RLA control RLA OLZ A

* * * * * * *

*

0 50 100 150 200 250

-7 0 7

% of baseline

Time (days)

Running wheel activity RHA

RHA control RHA OLZ

* * * ** ** ** ** ** ** ** ** ** **

B

Figure 14. Daily running wheel activity of RLA and RHA rats. Data are expressed as % of baseline activity. A: Daily running wheel activity of control and olanzapine treated RLA rats. B: Daily running wheel activity of control and olanzapine treated RHA rats. OLZ= olanzapine. *Significant difference versus control group (P<0.05). **Significant difference versus control group (P<0.01). Data shown represent mean ± s.e.m.

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28 ANOVA, RLA P<0.01; one-way ANOVA, RHA P<0.05). In addition, RLA control animals were significantly

more active in the dark phase compared to RHA control animals (one-way ANOVA, P<0.05) this difference was not present in the light phase. Furthermore, during the light phase OLZ treated animals seemed to be slightly more active than controls.

3.2.7 Body composition

On the last day of the experiment body composition was determined (see table 1). RLA rats seemed to have higher BW on day 14 compared to RHA rats but this difference was not significant. Lean body mass (LBM) of RHA control rats was significantly heavier than RLA control rats (one-way ANOVA, P<0.01).

Total fat mass of RLA control rats was significantly increased compared to RHA control rats (one-way ANOVA, P<0.05). In this study visceral fat was defined as the sum of peritoneal, retroperitoneal and gastro-intestinal tract adipose tissue. RLA control rats had significantly more visceral fat compared to RHA control rats (one-way ANOVA, P<0.05). Difference between skin in RLA control and RHA control failed to reach significance. Comparing brown adipose tissue (BAT) RLA control rats had significantly less BAT as compared to RHA control rats (one-way ANOVA, P< 0.01).

When comparing RLA OLZ and RHA OLZ rats with each other, RLA OLZ had significantly lower lean body mass and significantly less BAT (one-way ANOVA, P<0.05; one-way ANOVA, P< 0.01 respectively).

0 2000 4000 6000 8000

Dark Light

Revolutions

Activity Dark/Light

** *

†

Figure 15. Running wheel activity (revolutions) of control and olanzapine treated RLA and control and olanzapine treated RHA rats. Results are separated on dark phase and light phase. OLZ= olanzapine. *Significant difference versus control group (P<0.05). ** Significant difference versus control group (P<0.01).† Significant difference RLA control versus RHA control (P<0.05). Data shown represent mean ± s.e.m.

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29 There were no differences between RLA control and OLZ treated animals. When comparing RHA control

with OLZ statistical analysis did show significant differences. Although LBM just failed to reach significance (one-way ANOVA, F1,11= 4.879, P=0.052), OLZ treated RHA rats had significantly increased total fat compared to their controls (one-way ANOVA, P<0.01) and increased visceral fat (one-way ANOVA, P<0.05). Furthermore skin of RHA OLZ animals weighed significantly more than skin of RHA control rats (one-way ANOVA, P<0.05).

Table 1: Body fat distribution of control and olanzapine treated RLA and RHA rats.

a indicates a significant difference with RLA control rats (P<0.01). ^bindicates a significant difference with RLA control rats (P<0.05). ^c indicates a significant difference with RHA OLZ rats (P<0.01).^d indicates a significant difference with RHA control (P<0.05). ^eindicates a significant difference with RHA control (P<0.01). ^findicates a significant difference with RHA control (P<0.05). OLZ= olanzapine. Visceral fat= sum of peritoneal, retroperitoneal and gastro-intestinal tract adipose tissue. BAT=

brown adipose tissue.

RLA Control RLA OLZ RHA Control RHA OLZ Body weight (g) 288 ± 8.56 279 ± 7.70 277 ± 7.54 270 ± 4.20 Lean body mass (%) 43 ± 0.46 42 ± 0.62^d 46 ± 0.53^a 44 ± 0.55 Total fat (%) 12.8 ± 1.22 12.1 ± 1.08 8.9 ± 0.51^b 11.6 ± 0.38^e Visceral fat (%) 5.6 ± 0.45 5.1 ± 0.43 4.3 ± 0.30^b 5.4 ± 0.20^f

Skin (%) 16.6 ± 0.59 16.5 ± 0.76 15.1 ± 0.34 16.4 ± 0.34^f

BAT (%) 0.11 ± 0.01 0.13 ± 0.01^c 0.18 ± 0.01^a 0.18 ± 0.01

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30

3.3 Project 3: Olanzapine in RLA and RHA rats in a sedentary environment

Fig 16A and B display the change in BW from day 0, in RLA and RHA rats under control conditions and with OLZ treatment. In contrast to project 2, the animals in this project were in a sedentary environment (no running wheel).

-20 -15 -10 -5 0 5 10 15 20 25 30

-7 0 7

Gain (g)

Time (days)

Δ body weight RLA

-20 -15 -10 -5 0 5 10 15 20 25 30

-7 0 7

Gain (g)

Time (days)

Δ Body Weight RHA

RHA Control RHA OLZ

* * B

Figure 16. Delta body weight gain of female RLA and female RHA rats. A: delta body weight gain of control and olanzapine treated RLA rats. B: delta body weight gain of control and olanzapine treated RHA rats.

OLZ= olanzapine. *Significant difference versus control group (P<0.05). Data shown represent mean ± s.e.m.

*

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31 During this project there was no significant difference between control and OLZ treated RLA rats. At day

0 BW of RLA control animals was 285.5 ± 7.7 g and BW of RLA OLZ animals was 283.0 ± 6.6 g. From the start of the experimental period until day 7 both control and OLZ animals showed a steep increase in BW. After this increase control animals stabilized their body gain from day 8 at approximately 15 g. OLZ animals stabilized from day 9 at approximately 22.5 g gain compared to day 0. Furthermore both groups displayed large standard errors.

Baseline measurements between RHA control and RHA OLZ rats was similar. On day 0 BW of control animals was 267.6 ± 10.2 g and BW of OLZ animals was 259.7 ± 10.9 g. At the start of the experiment OLZ treated animals showed significantly lower weight gain for three days compared to the control animals (one-way ANOVA, P<0.05). From day 4 this difference disappeared. BW of RHA control and OLZ animals remained the same throughout the rest of the experimental period.

3.3.2 Food intake

Figure 17A and B show the daily FI of RLA and RHA rats under control conditions and with OLZ treatment. During baseline conditions both RLA control and OLZ and RHA control and OLZ rats had comparable FI. At day -1 RLA and RHA rats rapidly increased their FI on medium fat diet. During the experimental period there seemed to be a gradual decrease in FI of RLA rats. There also seemed to be a trend in which RLA OLZ animals had a slightly higher caloric intake compared to control animals. Looking at the experimental period of RHA rats there were no differences in FI.

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32 3.3.3 Water intake

In baseline period, WI of RHA control rats was significantly lower compared to RLA control rats (one-way ANOVA, P<0.01) (fig. 18). WI during week 1 and week 2 was significantly lower compared to baseline in all groups (paired samples t-test, P<0.05). There were no differences in WI between control and OLZ treated animals for both weeks.

0 20 40 60 80 100 120

-7 0 7

Food Intkae (kCal)

TIme (days)

Caloric Intake RHA

RHA Control RHA OLZ

0 20 40 60 80 100 120

-7 0 7

Food Intkae (kCal)

TIme (days)

Caloric Intake RLA

RLA Control RLA OLZ

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33 3.3.2 Glucose

Glucose responses to an IVGTT in RLA and RHA rats are shown in figure 19A and B. OLZ treatment did not affect glucose responses of RLA and RHA rats. Baseline blood glucose levels started at 5.85 ± 0.36 mM for RLA control and 6.06 ± 0.37 mM for RLA OLZ animals. Blood glucose levels increased to a maximum of 14.38 ± 2.83 mM for RLA control and 13.04 ± 0.83 mM for RLA OLZ treated animals during glucose infusion.

RHA control rats started with a blood glucose level of 5.96 ± 0.24 mM whereas RHA OLZ rats have basal glucose levels of 4.78 ± 0.50 mM. During glucose infusion RHA control animals increased their blood glucose levels to a maximum of 13.81 ± 1.64 mM. RHA OLZ animals had maximum glucose levels of 14.29 ± 1.53 mM. After termination of infusion, glucose levels of all groups turned to baseline within 30 minutes.

0 100 200 300

baseline week 1 week 2

Water (mL)

Cumulative Water intake

† ◊

Figure 18. Cumulative water intake of control and olanzapine treated female RLA and control and olanzapine treated female RHA rats. Results are separated into 3 weeks. OLZ= olanzapine. † Significant difference of RLA control versus RHA control versus control group (P<0.01). ◊ Significant difference versus baseline (P<0.05). Data shown represent mean ± s.e.m.

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34 3.3.3 Insulin

Insulin response of sedentary RLA en RHA animals are shown in figure 20 A and B. Statistical analysis did not show significant effects between control and OLZ treated animals for both RLA and RHA rats. Plasma insulin levels of RLA control animals started at 1.18 ± 0.32 mM and insulin levels of RLA OLZ started at 1.32 ± 0.28 mM. In addition, insulin levels increased to a maximum of 10.29 ± 3.16 ng/ml for RLA control

0 8 16

-60 0 60 120

Glucose (mM)

Time (min)

Glucose response RLA

0 8 16

-60 0 60 120

Glucose (mM)

Time (min)

Glucose response RHA

Figure 19. Glucose response to an IVGTT in RLA and RHA rats. A: Glucose response of control and olanzapine treated RLA rats. B: Glucose response of control and olanzapine treated RHA rats. OLZ= olanzapine. Data shown represent mean ± s.e.m.

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35 and 11.27 ± 2.43 ng/ml for RLA OLZ treated animals.

RHA control animals started with plasma insulin levels of 0.59 ± 0.22 mM and RHA OLZ had baseline insulin of 0.65 ± 0.30 mM. Remarkably, during glucose infusion insulin levels of RHA OLZ increased to a maximum of only 5.49 ± 1.10 ng/ml whereas RHA control animals showed maximum insulin levels of 7.91 ± 1.56 ng/ml. However, this was not a significant difference.

Comparing OLZ treated RLA and RHA animals using ANOVA showed significantly lower insulin levels for RHA OLZ animals on time point 0 and 10 (one-way ANOVA, P<0.05). On time point 5, the lower insulin levels of RHA OLZ animals compared to RLA OLZ just failed to reach significance (one-way ANOVA, F1,10=4.887, P=0.054).

0 4 8 12

-60 0 60 120

Insulin (ng/ml)

Time (min)

Insulin response RLA

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36 3.3.5 Activity

Cage activity of sedentary RLA and RHA rats expressed as percentage of baseline are shown in figure 21 A and B (for raw activity data see appendix fig. 2). Statistical analysis showed a significant difference during the entire project between RLA control and OLZ group and between RHA control and OLZ group (repeated measured, F1,10= 23.334, P<0.01; repeated measures, F1,7= 116.880, P<0.0001 respectively).

During baseline period control and OLZ treated animals of both RLA and RHA did not differ in cage activity. From the start of the experimental period RLA OLZ treated rats displayed significant lower activity levels compared to their controls with exception of day 7, 8 and 9 (one-way ANOVA, P<0.01).

RHA OLZ animals showed significantly lower cage activity every day following drug treatment (one-way ANOVA, P<0.01). Control RLA and RHA rats had activity levels in the experimental period that did not differ with their baseline activity. When comparing RLA control with RHA control animals, statistical analysis showed a significant decreased cage activity for RLA control rats (repeated measures, F1,8= 7.850, P<0.05). OLZ treatment abolished this difference.

0 4 8 12

-60 0 60 120

Insulin (ng/ml)

Time (min)

Insulin response RHA

Figure 20. Insulin response to an IVGTT in RLA and RHA rats. A: Insulin response of control and olanzapine treated RLA rats.

B: Insulin response of control and olanzapine treated RHA rats. OLZ= olanzapine. Data shown represent mean ± s.e.m.

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37 Cage activity during dark and light phase of the experimental period is shown in figure 22. Control

animals were most active during dark phase, whereas during light phase their activity levels decreased to approximately 30%. In dark phase OLZ treated rats showed significant decreased activity levels compared to their controls (paired samples t-test, P<0.0001). However, during light phase OLZ rats increased their activity significantly compared to their controls (paired samples t-test, P<0.0001).

0 20 40 60 80 100 120 140

-7 0 7

% of baseline

Time (days)

Activity RLA

RLA control RLA OLZ A

* * * * * * * * * *

*

0 20 40 60 80 100 120 140

-7 0 7

% of baseline

Time (days)

Activity RHA

RHA control RHA OLZ B

* * * * * * * * * * * * *

*

Figure 21. Daily cage activity of RLA and RHA rats. Data are expressed as % of baseline activity. A: Daily cage activity of control and olanzapine treated RLA rats. B: Daily cage activity of control and olanzapine treated RHA rats. OLZ= olanzapine.

Student Jacolien C. Graver (1726064)

Olanzapine in roman rats

Effects of olanzapine on body weight, adiposity and insulin sensitivity in female Wistar, Roman Low Avoidance and Roman High Avoidance rats.

Table of Contents

Abstract

List of abbreviations

1 Introduction

1.1 OLZ - side effects

1.2 OLZ – Pharmacology

1.3 OLZ - Gender differences

1.4 Animal model

1.5 Roman avoidance rats

1.6 Research aim

2 Materials & Methods

2.1 Animals

2.2 Surgery

2.3 Experimental design

2.4 Drug

2.5 Circadian registration

2.6 Intravenous glucose tolerance test

2.7 Post mortem analysis

2.8 Data analysis

3 Results

3.1 Project 1: Olanzapine in Wistar rats

∆ Body Weight

Daily Caloric Intake

Cumulative food intake

Daily Water Intake

Glucose Response

Insulin

Adiposity

3.2 Project 2: Olanzapine in RLA and RHA rats in an active environment

∆ Body weight RLA

∆ Body weight RHA

Daily Food Intake RLA

Daily Food Intake RHA

Cumulative Food Intake

Cumulative Water Intake

Glucose Response RLA

Glucose Response RHA

Insulin response RLA

Insulin response RHA

Running wheel activity

Running wheel activity RLA

Running wheel activity RHA

Activity Dark/Light

3.3 Project 3: Olanzapine in RLA and RHA rats in a sedentary environment

Δ body weight RLA

Δ Body Weight RHA

Caloric Intake RHA

Caloric Intake RLA

Cumulative Water intake

Glucose response RLA

Glucose response RHA

Insulin response RLA

Insulin response RHA

Activity RLA

Activity RHA