Insulin resistance following continuous, chronic olanzapine treatment: An animal model

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Schizophrenia Research 104 (2008) 23 – 30 www.elsevier.com/locate/schres

Insulin resistance following continuous, chronic olanzapine treatment: An animal model Araba F. Chintoh a,b , Steve W. Mann a,b , Tony K.T. Lam c , Adria Giacca a,c , Gary Remington a,b,d,⁎ a

d

Institute of Medical Science, University of Toronto, 1 King's College Circle, Toronto, Ontario, Canada M5S 1A8 b Centre for Addiction and Mental Health, 250 College Street, Toronto, Ontario, Canada M5T 1R8 c Department of Physiology, University of Toronto, 1 King's College Circle, Toronto, Ontario, Canada M5S 1A8 Department of Psychiatry, University of Toronto, 250 College Street, Rm 835A,Toronto, Ontario, Canada M5T 1R8 Received 24 February 2008; received in revised form 27 May 2008; accepted 4 June 2008

Abstract Some atypical antipsychotics have been linked to an increased propensity for weight gain and metabolic disturbances, including type II diabetes. The objective of this study was to investigate an animal model to help understand the mechanisms underlying this phenomenon. Female, Sprague–Dawley rats were treated with olanzapine (2.0 or 7.5 mg/kg, via osmotic mini-pump) for 4 weeks, followed by the hyperinsulinemic/euglycemic and hyperglycemic clamp procedures to assess insulin sensitivity and secretion in vivo. Changes in body weight, visceral fat, food intake and locomotor activity were also assessed. Hepatic glucose production (RA) was increased in the hyperinsulinemic/euglycemic clamp for both treatment groups compared to control rats, while the high-dose olanzapine group had decreased peripheral glucose utilization (RD). No changes in insulin secretion were detected in the hyperglycemic clamp. Olanzapine did not change body weight or food intake, but did result in significant accumulation of visceral fat and decreases in locomotor activity. Like others, we found that a rodent model for antipsychotic-related weight gain per se is not tenable. However, chronic treatment with olanzapine was found to confer both hepatic and peripheral insulin resistance independent of weight gain, indicating a direct effect on glucose dysregulation. © 2008 Elsevier B.V. All rights reserved. Keywords: Atypical antipsychotics; Olanzapine; Insulin resistance; Weight; Osmotic mini-pump

1. Introduction Certain atypical antipsychotics have been linked to an increased risk of weight gain and metabolic disturbances compared to their conventional counter⁎ Corresponding author. Centre for Addiction and Mental Health, 250 College Street, Toronto, ON, Canada M5T 1R8. Tel.: +1 416 535 8501x6044; fax: +1 416 979 4292. E-mail address: [email protected] (G. Remington). 0920-9964/$ - see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.schres.2008.06.006

parts (Henderson et al., 2000; Newcomer et al., 2002; Newcomer, 2004; Zipursky et al., 2005; Petersen and McGuire, 2005; Sernyak et al., 2005). The weight gain is both rapid and substantial, although liability differs considerably between the different atypical agents. Risk of glucose dysregulation is highest with the atypical compounds associated with the greatest risk of weight gain i.e., clozapine, olanzapine, giving rise to the notion that it is the weight gain that accounts for the metabolic abnormalities (Allison et al., 1999). However, evidence

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of acute metabolic effects of olanzapine (Houseknecht et al., 2007) as well as report of reports of diabetic ketoacidosis soon after the onset of treatment, and in individuals not manifesting notable weight gain (Rigalleau et al., 2000; Selva and Scott, 2001; Ramankutty, 2002; Masand et al., 2005), suggests that the drugs in and of themselves may influence glucose dysregulation. Animal models represent a means to more closely examine these issues (Goudie et al., 2002; Melkersson, 2004; Fell et al., 2005). The present study was carried out to evaluate the effects of chronic olanzapine administration on body weight, food intake, visceral fat, locomotor activity and glucose regulation (specifically insulin sensitivity and secretion) in a rodent model. 2. Methods and materials 2.1. Animals Female, Sprague Dawley rats (Harlan, Indianapolis, IN) weighing 225–275 g were singly housed in plastic cages and maintained on a 12 h light/dark cycle (lights on at 6: 00am) at 22 °C and 50% humidity. Food (standard rodent chow, Harlan Teklad) and water were available ad libitum. Animals were treated in compliance with the guidelines of the Canadian Council on Animal Care, and the University of Toronto Animal Care Committee approved all protocols. 2.2. Drugs Animals were treated with either 2.0 or 7.5 mg/kg of olanzapine (Toronto Research Chemicals, Toronto, Canada) dissolved in 1% acetic acid and administered via an Alzet® osmotic mini-pump (Alzet 28-day model 2LM4, Durect Corporation, Cupertino, California) implanted subcutaneously through a 2 cm incision between the scapulae. The incision was closed with surgical staples and the animal was returned to its heated home cage to recover. Each osmotic pump holds approximately 2 mL and is primed to infuse at a rate of 2.5 μL/h delivering 60 μL/day for 28 days. Various lines of investigations influenced choice of drug, dose, and route of administration. Clinically, olanzapine represents one of the atypical antipsychotics with greatest risk of inducing weight gain and/or metabolic disturbances (Allison et al., 1999; Zipursky et al., 2005; Wu et al., 2006), and we wanted to maximize the chance of observing a treatment effect. The doses chosen were selected to reflect what is observed in the clinical setting based on a) plasma levels (recognizing the marked variability reported in plas-

ma levels and lack of a ‘therapeutic window') (Perry et al., 2001); and, b) Positron Emission Tomography (PET) findings i.e., 70% D2 receptor occupancy, a threshold in humans associated with optimal chance of clinical response (Kapur et al., 2003; Kapur and Remington 1996; Kapur and Remington, 2001). Using this approach, two doses were employed: 2.5 mg/kg based on plasma levels and 7.5 mg/kg based on central D2 occupancy (hereafter referred to as low-dose and high-dose, respectively). The choice of administration by osmotic pump acknowledged the rapid metabolism of antipsychotics reported in rodents (Chiu and Franklin, 1996), and an effort to more closely approximate the human condition by providing sustained exposure. In addition, female animals were studied because preexisting literature suggests female rats demonstrate greater obesigenic effects of atypical antipsychotics than males (Goudie et al., 2002; Minet-Ringuet et al., 2005). 2.3. Procedures 2.3.1. Activity monitoring Body weight and food intake were measured weekly. Locomotor activity was determined by placing the rats in an activity monitoring box for 120 min. Locomotion was recorded at 5-minute intervals via 6 photobeam cells placed 3 cm above the floor of the activity box. Upon completion of the study, rats were sacrificed with an overdose of ketamine hydrochloride. Visceral fat from the omentum was removed and weighed post-mortem. Omental fat depots were chosen to reflect the clinical condition, as patients with antipsychotic-induced weight gain develop a central distribution of adipose tissue and insulin resistance is known to occur at the site of this visceral fat (Stolic et al., 2002). 2.3.2. Surgical procedures After 25 days of drug treatment, animals were anaesthetized with isofluorane and administered buprenorphine for post-operative analgesia. Polyethylene catheters (PE-50, Cay Adams, Boston, MA) with 2.5 cm of silastic tubing (Dow Corning Corp., Midland, MI) were introduced and advanced to the right atrium and aortic arch in the jugular vein and carotid artery, respectively. The catheter lines were externalized dorsally and blocked with a pin. 2.3.3. Hyperinsulinemic–euglycemic/hyperglycemic clamp To assess change in glucose control, we used the twostage euglycemic–hyperinsulinemic/hyperglycemic

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clamp procedure. These clamp methods are considered the gold standard for assessing insulin sensitivity and secretory functioning of the pancreatic beta cells in vivo (Uwaifo et al., 2002). The euglycemic clamp technique allows for the determination of whole whole-body insulin sensitivity; subjects are infused with insulin as well as a variable rate of exogenous glucose to counteract the insulin-induced decline in plasma glucose levels. Use of radioactive tracer helps determine rates of hepatic glucose production and peripheral glucose uptake. The hyperglycemic clamp provides an index of the secretory capacity of pancreatic beta cells, and can assess sensitivity of peripheral tissues (Elahi, 1996). Subjects are given an initial bolus of glucose to elevate plasma glucose rapidly, and then a fixed degree of hyperglycemia is maintained via infusion of exogenous glucose. Plasma is sampled frequently for levels of insulin and C-peptide. The hallmark of this technique is subjects' unmitigated exposure to the same concentration of glucose, facilitating the evaluation of in vivo beta cell response to a glucose challenge. Animals were allowed 3 days to recover from surgery, and after an overnight fast catheter lines were extended to connect with the infusion pumps (see Fig. 1 for a schematic of the experimental protocol). Radioactive tracer ([H3]-glucose, 20 μCi) was infused for 90 min, followed by infusion of insulin (5 mU/kg/min) at a rate of 10 μL/min. Euglycemia was maintained via exogenous glucose given at a variable rate according to plasma

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glucose levels determined every 5 min. The exogenous glucose was labeled (specific activity = 48 μCi/g) to maintain plasma glucose specific activity constant at basal level. Plasma samples were collected every 10 min during the 30 min tracer equilibration period before the clamp, during the final 30 min of the insulin challenge. After 120 min of recovery time the hyperglycemic clamp was initiated with a glucose bolus (500 mg/kg) infused into the jugular catheter. Variable infusion of exogenous glucose maintained plasma glucose levels at approximately 300 mg/dL for an additional 90 min. Plasma samples were collected throughout this clamp for insulin and C-peptide assay. 2.4. Laboratory methods Plasma glucose was measured with a Beckman Glucose Analyzer II (Beckman, Fullerton, CA). Plasma radioactivity from [3-3H] glucose was determined after deproteinization with Ba(OH)2 and ZnSO4, and subsequent evaporation to remove tritiated water. Aliquots of the [3-3H] glucose and of the tritiated glucose infusate were assayed together with the plasma samples. The intra-assay coefficient of variation was 2.5%, and the inter-assay coefficient of variation was 6.5%. Insulin and C-peptide levels in plasma were determined by radioimmunoassay using kits specific for rat insulin (100% cross-reactivity with porcine insulin used for infusion) and C-peptide from Linco

Fig. 1. Experimental protocol describing the hyperinsulinemic + hyperglycemic clamps carried out after 4-week treatment with high-dose olanzapine (7.5 mg kg− 1 day− 1) or low-dose (2 mg kg− 1 day− 1) olanzapine or vehicle control given by continuous s.c. delivery via osmotic pumps to female Sprague Dawley rats fed standard chow.

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Fig. 2. Effect of 4-week treatment with low-dose (2 mg kg− 1 day− 1) or high-dose olanzapine (7.5 mg kg− 1 day− 1) (low-ola and high-ola respectively) given by continuous s.c. delivery via osmotic pumps on body weight gain (A), visceral (mesenteric and omental) fat (B), food intake (C) and activity level (D) in female Sprague Dawley rats fed standard chow. High-dose olanzapine increased visceral fat, and both high-dose and low-dose olanzapine decreased activity levels. ⁎P b 0.05 high-ola vs. control and low-ola. ⁎⁎P b 0.001 low and high-ola vs. control.

Research (St. Charles, MO). The coefficients of variation were b9 and 10.5% for insulin and C-peptide, respectively. Plasma FFA levels were measured using a colorimetric kit from Wako Industrials (Osaka, Japan). Glucose infusion rate (GIR) was derived based on each animal's weight and glucose infusion pump rate. Glucose turnover (rate of appearance, RA, of glucose determined with [3-3H] glucose) was calculated using steady-state formulae (Stetten et al., 1951), taking into account the extra tracer infused with the glucose infusate (Finegood et al., 1987). In the basal state, the total rate of glucose appearance corresponds to endogenous glucose production. During the clamps, endogenous glucose production was calculated by subtracting the exogenous glucose infusion rate from the total rate of glucose appearance. At steady state, glucose disappearance, RD, corresponds to the rate of glucose appearance, and at euglycemia glucose disappearance corresponds to tissue glucose utilization because renal glucose clearance is zero. Data are presented as average values of samples taken between 60–90 min and 150–180 min of the hyperinsulinemic–euglycemic clamp experiment.

Though the observed increases in body weight were marginal, chronic olanzapine had a significant effect on total visceral fat, Fig. 2B. The high-dose group had a significant increase in visceral fat, 3.2 ± 0.4 g compared to the low-dose (1.8 ± 0.2 g) and control animals (1.4 ± 0.2 g), P b 0.05.

2.5. Statistical analysis

3.3. Food intake

T-tests or ANOVA for repeated measurements were performed using Statistica software (StatSoft Inc., Tulsa, OK). Significance was accepted at P ≤ 0.05.

Olanzapine treatment had a moderate effect on food intake, once again in a dose-dependent fashion, Fig. 2C. Specifically, the high-dose group consumed 20.4 ± 0.5 g/

3. Results 3.1. Body weight After 4 weeks of treatment, the high-dose group had a total weight gain of 32.8 ± 3.2 g (13.5 ± 1.3% of their baseline weight), the low-dose group 29.7 ± 3.1 g (12.2 ± 1.3% of their baseline weight), and the controls 29.3 ± 3.9 g (12.2 ± 1.7% of their baseline weight). Though the olanzapine-treated animals demonstrated the greatest weight gain in a dose-dependent fashion, these differences were not significant, P = 0.74, Fig. 2A. 3.2. Visceral fat

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day of standard chow, compared to the low-dose and control animals who consumed 19.6 ± 0.2 g/day and 19.0 ± 0.5 g/day, respectively. However, these differences were not significant, P = 0.08. 3.4. Locomotor activity The baseline activity level of each group did not differ, P = 0.4. After 1 week of treatment, both the high- and low-dose groups were significantly less active, P b 0.001. This decrease in locomotor activity was sustained throughout the entire duration of the treatment period (total locomotor activity counts: control = 862 ± 48, low-dose = 466 ± 30, high-dose = 427 ± 29, P b 0.001), Fig. 2D. 3.5. Euglycemic clamp 3.5.1. Basal period Glucose levels between all groups were similar during the basal period (control: 127 ± 2.27 mg/dL; lowdose: 126 ± 2.18 mg/dL; high-dose: 130 ± 1.46 mg/dL) (data not shown), P = 0.75. In addition, there were no differences between levels of glucose production, glu-

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cose utilization or free fatty acids (FFAs; data not shown) between 60 and 90 min, P N 0.4. 3.5.2. Steady state Similar glucose levels were maintained in each group, P = 0.1 (data not shown). Chronic administration of highdose olanzapine decreased the GIR, Fig. 3A. Glucose infusion rates for the control group were significantly higher than those for the high-dose group, P = 0.01. The GIR for the low-dose group did not differ from that of control animals, P = 0.6. Mean glucose production, RA, was 6± 3 μmol/kg min for control animals and was increased in the high-dose (20 ±3 μmol/kg min, P = 0.06) and low-dose animals (21± 3 3 μmol/kg min, P = 0.03), Fig. 3B. Glucose utilization, RD, was lower in only the low-dose rats (143 ± 5 μmol/kg min compared to vehicle 164 ± 3 μmol/kg min, P = 0.02). The RD for the high-dose group (172 ± 5 μmol/kg min) did not differ from the control group, P = 0.08, Fig. 3C. Plasma insulin levels in the low-dose group (795 ± 68 pM) also differed significantly from controls (571 ± 24 pM), P = 0.03, although this was not the case for high-dose animals vs. controls, P = 0.2, Fig. 3D. FFA levels remained unchanged, P = 0.4.

Fig. 3. Effect of 4-week treatment with low-dose (2 mg kg− 1 day− 1) or high-dose olanzapine (7.5 mg kg− 1 day− 1) given by continuous s.c. delivery via osmotic pumps on glucose infusion rate (A), glucose production (B), glucose utilization (C) and insulin levels (D) during the hyperinsulinemic euglycemic clamp described in Fig. 1. High-dose olanzapine decreased the glucose infusion rate (i.e., induced insulin resistance) due to an increase in glucose production (hepatic insulin resistance) and a decrease in glucose utilization (peripheral insulin resistance). Low-dose olanzapine induced hepatic insulin resistance. The low-dose of olanzapine increased the insulin levels (i.e., decreased insulin clearance). a = P b 0.05 high-ola vs. control. b = P b 0.05 low-ola vs. control. c = P b 0.05 high-ola vs. low-ola. d = P b 0.05 low-ola vs. control.

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Fig. 4. Effect of 4-week treatment with low-dose (2 mg kg− 1 day− 1) or high-dose olanzapine (7.5 mg kg− 1 day− 1) given by continuous s.c. delivery via osmotic pumps on plasma glucose levels (A), glucose infusion rate (B), plasma insulin (C) and C-peptide levels (D) during the hyperglycemic clamp. There was no significant difference in both insulin and C-peptide secretory response among groups.

3.6. Hyperglycemic clamp 3.6.1. Basal period Basal glucose levels were comparable between the groups, Fig. 4A. Plasma insulin and C-peptide levels did not differ, P N 0.2 (Fig. 4C, D). 3.6.2. Glucose challenge Plasma glucose for each group was maintained at approximately 17 mM. There was no significant difference in either the insulin (P = 0.5) or C-peptide (P = 0.3) secretory response among groups, (Fig. 4A–D). 4. Discussion Olanzapine was not associated with significant weight gain, a key feature of this drug's profile when administered to humans. This was not entirely surprising, as the validity of a rodent model for antipsychoticrelated weight gain has been challenged elsewhere (Baptista et al., 2002). Several other findings, however, suggest that it may be premature to dismiss the model categorically. An increase in visceral fat, which in turn can be linked to insulin resistance, was observed with the higher dose of olanzapine. Increased visceral adiposity is commonly described as part of the clinical picture in antipsychotic-

related weight gain in humans (Bergman and Ader, 2005; Faulkner et al., 2007), suggesting that the model is not entirely without merit in mirroring what occurs in humans. Where this model may prove particularly useful is in evaluating the putative direct effect of these medications, independent of weight gain. In both the low- and highdose olanzapine groups, there was no significant weight gain and in the former no changes in visceral fat as well. Despite this, both groups demonstrated significant effects on glucose regulation, compelling evidence for a possible direct effect related to the medications themselves. A recent report from Houseknecht et al. (2007) provides evidence that olanzapine can have an immediate effect on insulin sensitivity. After acute treatment with olanzapine, they observed a reduction in GIR within 100 min. These novel data are in line with our findings which highlight the ability of olanzapine to alter metabolism independent of changes in body weight. At endpoint the high-dose group demonstrated significantly lower GIR requirements to maintain euglycemia, indicating an adverse effect on whole-body insulin sensitivity. Both high- and low-dose treated rats exhibited more than a two-fold increase in glucose production over control animals. High-dose animals also exhibited significant peripheral insulin resistance. These data suggest olanzapine suppresses the insulin-induced inhibition of glucose production and diminishes the

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ability of insulin to facilitate uptake of glucose into muscle cells. Low-dose animals also exhibited higher insulin levels than the control group, reflecting a deficit in insulin clearance. In the hyperglycemic clamp, there were no significant differences in GIR. Assay analysis did not reveal any differences between the insulin and C-peptide levels. This lack of effect is surprising in light of the clinical reports of hyperinsulinemia, but may reflect adaptation to the drug over the course of the 4-week treatment period. Also, the stress of the prolonged experiments and the residual influence of the hyperglycemic clamps (i.e., glucose toxicity) might have masked drug-related differences although results from Ader et al. (2005) in dogs corroborate the present findings. In addition, data investigating the role of the cholinergic system in insulin secretion also highlight an inability of olanzapine to impair glucose-stimulated insulin secretion. Johnson et al. (2005) investigated the impact of olanzapine and other atypicals on rodent pancreatic islet cells. Olanzapine impaired insulin secretion in cells bathed with both carbachol and glucose but not with glucose alone. As olanzapine is a potent antagonist of the muscarinic M3 receptor (Richelson, 1999), the authors concluded olanzapine impairs insulin secretion selectively in the presence of carbachol because of its ability to block the M3 receptor. It is thought, however, that the cholinergic system is bypassed when islet cells are stimulated by intravenous infusion of glucose (Johnson et al., 2005). If olanzapine does impair pancreatic islets through its action on the muscarinic receptor then it is not surprising that olanzapine did not effect levels of insulin and Cpeptide in the present study, as insulin secretion was stimulated by an intravenous glucose bolus. Several behavioral measures i.e., food intake, locomotor activity, were incorporated to better understand the mechanisms by which weight gain occurs. Interestingly, there was no change in food intake as a function of drug treatment, although a significant decrease in locomotor activity was noted within the first week of treatment for both olanzapine groups compared to control. Clozapine and olanzapine, the two atypicals linked to the greatest risk of weight gain are also characterized by prominent sedation (Casey, 1996; Richelson, 1999), which may translate to significant activity reduction and resultant weight gain in humans even without changes in diet or total intake. The implications of the present findings here are, of course, tempered by the lack of significant weight gain in the olanzapinetreated animals. In summary, using a gold standard method for assessing insulin sensitivity, we have successfully demon-

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strated in an animal model that chronic and sustained administration of olanzapine effects insulin resistance through both hepatic and peripheral mechanisms. Evidence that olanzapine can elicit these changes without a simultaneous increase in body weight may represent an advantageous finding, as it permits the evaluation of the immediate impact of antipsychotics glucose regulation independent of the confounding influence of weight gain. Role of funding source In the last 5 years Gary Remington has received grant support from the National Alliance for Research in Schizophrenia and Depression (NARSAD), the Stanley Medical Research Institute, Merck (Germany) and Novartis. These sources had no further role in study design, collection, analysis and interpretation of data, in the writing of the report or in the decision to submit the paper for publication. Contributors Each of the authors made a significant contribution to the study design, data collection, data analysis and/or manuscript preparation. All authors have approved the final manuscript. Conflict of interest The authors declare they have no conflicts of interest with regard to the work presented in this report. Acknowledgements Some of the data presented in this article were previously published as an abstract in The International Journal of Neuropsychopharmacology, 2006. The authors would like to thank Ping Han, Loretta Lam, Tony K. Lam and Anthony Nassan for their help with conducting the clamp experiments, collecting tissue and performing assays.

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