Clozapine is an atypical antipsychotic drug with unique pharmacological and therapeutic properties. Unlike the typical antipsychotic drug, haloperidol, clozapine does not cause extrapyramidal side effects; however, weight gain, dyslipidemia, and type II diabetes are commonly associated with the use of this drug in subjects with schizophrenia. The aim of this study was to profile gene expression in the rat striatum following clozapine treatment. Chronic treatment with clozapine revealed upregulation of several genes including the glucose-dependent insulinotropic polypeptide (GIP) gene by over 200% in the rat striatum. The cDNA array results for the GIP gene were confirmed by real-time RT-PCR as well as by radioimmunoassay. Expression of the GIP gene in the central nervous system is consistent with the results of retinal GIP gene expression as reported by other investigators. Taken together, these findings implicate the possible role of GIP as a neuromodulator in the central nervous system. GIP is an insulinotropic agent with stimulatory effects on insulin synthesis and release from the pancreas. However, changes in brain GIP levels are most likely unrelated to the metabolic adverse effects (dyslipidemia, type II diabetes, weight gain) associated with clozapine treatment. Therefore, we also measured GIP gene expression in the K-cell-rich regions, duodenum and jejunum (small intestine), and plasma GIP levels using radioimmunoassay following chronic treatment with clozapine. GIP mRNA levels in the small intestine and the plasma GIP at the protein level were significantly elevated in clozapine-treated subjects. Furthermore, as observed in humans, chronic clozapine treatment also caused weight gain, and increased levels of insulin, triglycerides and leptin in the plasma. These results suggest that adverse metabolic effects associated with clozapine treatment may be related to its ability to increase intestinal gene expression for GIP.
Clozapine belongs to the class of atypical antipsychotic drugs and has lower dopamine D2 receptor affinity relative to that of typical antipsychotic drugs such as haloperidol.1, 2, 3, 4, 5, 6, 7 At therapeutic doses, clozapine displays dopamine D2 receptor blockade ranging from 20 to 60% occupancy.4 It has a heterogeneous receptor binding profile that includes affinity for dopamine D1, D2 and D4, serotonin (5–HT) 5–HT2A, 5–HT2C and 5–HT3, muscarinic M1, histaminic H1 and α-adrenergic α1 and α2 receptors.5, 6, 7 In addition to treating positive symptoms of schizophrenia, clozapine has been shown to effectively treat secondary negative symptoms and cognitive deficits.8, 9 Clozapine has been shown to be successful in the treatment of schizophrenia within suicidal patients.10
Treatment with clozapine does not cause motor-related side effects or hyperprolactinemia which are serious adverse effects that occur following treatment with the majority of antipsychotic drugs. However, several metabolic side effects are associated with its use. These may include type II diabetes mellitus, weight gain and dyslipidemia.7, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 Type II, or non-insulin-dependent, diabetes mellitus has become one of the most undesirable side effects of atypical antipsychotic drugs. There have been a number of reported cases of type II diabetes mellitus in patients treated with clozapine. Hagg et al.12 conducted a case report that found that type II diabetes occurred in 12% of patients treated with clozapine compared to 6% of patients treated with typical antipsychotic drugs. Henderson et al.13 performed a 5-year naturalistic study of patients treated with clozapine and found that 36.6% of patients developed type II diabetes mellitus during the study period. In addition, Yazici et al. discovered that clozapine caused an increase in blood glucose and elevated insulin levels, and concluded that the impaired glucose tolerance was due to insulin resistance.18 These findings were recently substantiated by Henderson et al.22 Furthermore, Melkersson et al.15 found that patients treated with clozapine had insulin levels that were more frequently elevated than those treated with typical antipsychotic drugs, suggesting that clozapine might increase insulin secretion and induce insulin resistance.
Clozapine-induced type II diabetes mellitus is thought to be caused by several direct and indirect mechanisms. There are a number of theories with respect to clozapine-associated dysregulation of relevant metabolic pathways. Weight gain caused by atypical antipsychotic drugs may be a contributing factor to the glucose dysregulation. Weight gain accompanied by increases in adipose tissue volume can lead to insensitivity to insulin and glucose intolerance.17 Atypical antipsychotic drugs may decrease the concentration of insulin-sensitive glucose transporters (GLUT4) or may cause an inability to stimulate GLUT4 recruitment from microsomes due to elevated concentrations of glucose and insulin.23, 24 Furthermore, antagonism of the serotonin 5-HT1A receptor by atypical antipsychotic drugs may decrease pancreatic β-cell responsiveness to glucose levels that can lead to glucose dysregulation.17 However, the precise mechanisms by which clozapine causes adverse metabolic effects are unknown. Relative metabolic adverse effects caused by various atypical antipsychotic drugs are summarized in Table 1.
The aim of this study was to profile gene expression in the rat striatum following clozapine treatment. Using cDNA expression array technology, several genes were altered by chronic treatment with clozapine; specifically, (GIP) showed an increase of greater than 200%. The increase in the GIP gene was further investigated in relation to metabolic adverse effects caused by clozapine treatment.
cDNA expression array analysis reveals an increase in GIP mRNA expression following clozapine treatment
To identify novel genes that might be associated with clozapine's effects in the rat striatum, we compared gene expression between control and clozapine treated rats. The cDNA array results obtained following clozapine treatment indicate significant up- and down-regulation of several genes in the rat striatum and are shown in Figure 1, and variation among the cDNA arrays is shown in Table 2. The phosphorimaging signal of GIP is enlarged to provide a clearer representation of the differential gene expression (Figure 1a). GIP (Accession # L08831) was chosen for further analysis based on its putative involvement in the metabolic side effects caused by atypical antipsychotic drugs. Since the GIP gene has not been shown to be expressed in the brain, the array spot of GIP was first confirmed in this region by RT-PCR to rule out the possibility of false positives. Results displayed in Figure 2 show 100% homology of rat intestinal pro-GIP cDNA with the rat striatum (219 bp) PCR product. Furthermore, the sequence between intestinal mature, physiologically active GIP and striatal GIP is also 100% identical (see Figure 2). Presence of GIP in the human and rat striatum was further confirmed by an immunoprecipitation-mass spectrophotometry technique by Dr Wolf in Germany (unpublished results). Immunohistochemistry was also carried out to determine whether GIP is expressed in dopaminergic neurons along with tyrosine hydroxylase.28, 29 As shown in Figure 3, GIP is colocalized with tyrosine hydroxylase in presynaptic dopaminergic neurons.
Effects of clozapine on GIP gene expression in the rat striatum and small intestine
To confirm the cDNA array results, mRNA levels for GIP were determined in the striatum, and to relate clozapine's effects to metabolic disorders, intestinal GIP mRNA levels were also quantified. Clozapine treatment caused a significant increase of GIP gene expression in both the intestine (300%) and the striatum (100%). A representative real-time RT-PCR amplification plot is shown in Figure 4a and b for the small intestine and striatum, respectively. The fold change in the GIP gene is shown in Figure 5. The expression level of the housekeeping gene, cyclophilin, remained unchanged (Figure 5).
Effect of clozapine on plasma and striatal GIP concentrations
To establish whether increased gene expression results in an increase plasma and striatal GIP at the protein level, GIP concentrations were quantified by radioimmunoassay. The results displayed in Figure 6a and b show a significant (P<0.001) increase in GIP protein concentrations in both the plasma and striatal extracts, suggesting increased translation of the expressed gene in both the intestine and the striatum following chronic drug treatment.
Effect of clozapine on weight gain and hormones related to glucose homeostasis
To determine whether clozapine treatment is related to metabolic abnormalities, fasting plasma levels of glucose, insulin, triglycerides and leptin were quantitated at the end of the 28-day drug treatment period. As shown in Figure 7, significantly elevated insulin, glucose, triglycerides and leptin levels were found in the clozapine-treated subjects compared to the saline-treated controls. Clozapine-treated subjects also displayed significant increases in weight gain over saline controls (Figure 7).
cDNA expression array technology was utilized to profile gene expression alterations in the striatum following chronic clozapine treatment. GIP was chosen for further analysis in order to investigate its putative involvement in the metabolic side effects caused by atypical antipsychotic drugs and to examine its elevated expression levels in relation to its adverse metabolic effects. However, considering the fact that the GIP gene has not been previously shown to be expressed in the brain, and that the occurrence of false positives is a common problem in cDNA array technology due to spotting, pipetting and other errors, it was essential to establish its identity in this tissue before embarking upon further studies. Therefore, using rat intestinal GIP primers and RT-PCR, we amplified a fragment from the gene of the rat striatum. The 219 bp PCR product displayed 100% homology with the rat intestinal pro-GIP gene (Figure 2). The upregulation of the GIP gene's expression was confirmed by real-time RT-PCR and by radioimmunoassay in the striatum as well as in the small intestine and plasma (Figures 3, 4, 5).
GIP mRNA has not previously been detected in any regions of the brain, liver or pancreas. However, the GIP receptor has been shown to be expressed in the pancreatic islet β cells, stomach, small intestine, adrenal cortex, brain, pituitary and adipose tissue.30 Specifically, in the brain, the majority of the GIP receptor mRNA is found in areas that comprise the limbic system.30, 31 This study provides conclusive evidence for the expression of GIP in the brain. This finding is not unexpected since another incretin glucagon-like polypeptide, which is secreted from L cells of the small intestine, has also been shown to be expressed in the brain.32 Furthermore, a detailed regional distribution of GIP and GIP receptor mRNA expression in the human brain has been established.28, 29 In addition, colocalization of GIP with tyrosine hydroxylase indicates that GIP may play a neuromodulatory role in the brain (Figure 3). These results are consistent with the finding of Cho et al.,1 who demonstrated GIP expression at both the mRNA and protein level in rat retina. Collectively, these findings suggest GIP's role as a neuromodulator in the CNS.
GIP is one of the major incretins of the enteroinsular axis.33, 34, 35, 36, 37, 38 In the presence of elevated levels of glucose, GIP stimulates insulin secretion from pancreatic islet β-cells.39, 40 GIP is secreted into the blood from the K cells of the duodenal and jejunal mucosa, and travels through the bloodstream to the pancreatic islet β cells where it binds to its receptor, activating a heterotrimeric GS protein that stimulates adenylyl cyclase.30, 41, 42, 43, 44, 45 This results in an increase in cyclic adenosine monophosphate (cAMP), activation of protein kinase A (PKA) and subsequent increase in Ca2+ influx via voltage-gated Ca2+ channels. The increase in free Ca2+ concentration leads to insulin secretion.45, 46, 47
GIP has been linked to the onset of type II diabetes mellitus. In certain groups of patients, chronically high plasma GIP concentrations have been found and elevated levels of GIP have been shown in the retina of streptozotocin-induced diabetic rats.1, 48, 49, 50
In this study, we show that clozapine leads to glucose intolerance in rats. The levels of glucose, insulin and GIP remained significantly elevated over the saline-treated control group, suggesting that the antipsychotic drug leads to impairment in glucose effectiveness. These findings are consistent with human studies which showed that atypical antipsychotic drugs lead to glucose intolerance, high leptin levels and increased weight gain.7, 22, 51, 52
Although the precise mechanism of action of increased GIP secretion by clozapine cannot be explained by the results reported here, the hypothesized mechanism of clozapine-induced diabetes mellitus type II lies in its ability to antagonize the α2-adrenergic receptor. Stimulation of the α2-adrenergic system has been shown to significantly inhibit the GIP's response to glucose ingestion through a direct effect on pancreatic cells.53 The α2-adrenergic receptors are negatively coupled to the adenylyl cyclase system via the inhibitory Gi protein.54 Therefore, antagonism of α2-adrenergic receptors by clozapine may increase cAMP levels that may induce increased GIP gene expression by the induction of phospho-CAMP response element-binding protein (CREB) and consequent binding of this transcription factor to the GIP gene promoter, since the GIP gene promoter has two CREB-binding sites.55, 56 Clozapine has an affinity for multiple receptors: serotonergic, cholingergic, muscarinic, histaminergic and dopaminergic receptors, and the small intestine does express these receptors; therefore, an alternative causal mechanism involving these receptors with or without α-adrenergic receptors which leads to increased GIP gene expression by clozapine cannot be ruled out. Furthermore, plasma GIP levels are sensitive to meal intake, therefore, GIP levels in the clozapine-treated subject, along with insulin and glucose should be determined following a glucose tolerance test. In conclusion, the results reported in this study clearly show the role of GIP in clozapine-induced adverse metabolic effects and provide a conceptual framework for further studies in subjects with schizophrenia treated with atypical antipsychotic drugs such as clozapine and olanzapine.
Materials and methods
Animal housing and drug treatment
Sprague–Dawley rats (250–260 g) were purchased from Charles River Canada (St Constant, QC, Canada). Animals were housed and tested in compliance with the guidelines described in the Guide to Care and Use of Experimental Animals (Canadian Council on Animal Care, 1984, 1993). All animals were housed individually with food and water available ad libitum. The composition of food was as follows: carbohydrate 51.19%; crude protein, no less than 22.0%; crude fat, no less than 5.0%; crude fiber, no less than 4.5%; Vitamin A acetate; Vitamin D3 supplement; Vitamin E supplement; niacin; calcium pantothenate; riboflavin; thiamine mononitrate; pyridoxine hydrochloride; menadione; sodium bisulfite complex; folic acid; biotin; Vitamin B12 supplement; magnesium oxide; manganous oxide; ferrous sulfate; copper sulfate; zinc oxide; calcium iodate; cobalt carbonate and chromium potassium sulfate. The rats were divided into two equal groups: the clozapine group (n=3 for cDNA array study, n=10 for striatal real-time RT-PCR, n=10 for small intestine real-time RT-PCR and n=10 for radioimmunoassay) and the control group (n=3 for cDNA array study, n=10 for striatal real-time RT-PCR, n=10 for small intestine real-time RT-PCR and n=10 for radioimmunoassay). Clozapine (Sigma Aldrich, Oakville, ON, Canada) was administered 20 mg/kg/day by oral route (gavage) to mimic the human situation, while the animals in the control group received 0.9% saline at volume per weight amounts comparable to those of the clozapine. The dose of clozapine was determined on the basis of its therapeutic equivalent dose used in humans and adjusted for the faster metabolic rate of rodents. Clinical doses of clozapine in human subjects range from 200 to 800 mg/day,57 and the half-life of clozapine is roughly 3–4 h in rats, as compared to 24 h in humans.58 The drug was given orally by gavage between 1800 and 2000 hours for 28 consecutive days. The weights of the animals were recorded on a daily basis.
Animal dissection, tissue handling and storage
Following the final drug administration, the rats were anesthetized with isoflurane and 5 ml of fasting blood was collected from the heart in polypropylene tubes containing EDTA and then centrifuged at 1600g for 15 min to separate the plasma. The plasma was stored at −80°C until further use. The rats were decapitated, and striatal and small intestine (duodenum and jejunum) tissues were dissected out and stored at -80°C until further use.
Total RNA was isolated from the dissected striatal and small intestine tissues using Trizol reagent (Invitrogen-Life Technologies, Burlington, ON, Canada), and purity was checked as previously described.59
cDNA expression array probe preparation, hybridization and array analysis
The Atlas Rat 1.2 Array kit was used according to the manufacturer's protocol (Clontech, Palo Alto, CA, USA). The study was performed using the striatal samples from clozapine-treated (n = 3) and control-treated (n = 3) rats as described previously from our laboratory.59 Duplicate arrays were used per experimental and control samples. 32P-labelled cDNA probes were created from each RNA sample using Moloney Murine Leukemia Virus (MMLV) reverse transcriptase and primers specific for the gene sequences found on the cDNA expression arrays. Each array contained 1176 rat genes (http://www.clontech.com). Probes having specific activity in the range of 2 × 106–10 × 106 counts/min were used in the hybridization. To reduce non-specific binding and to facilitate probe hybridization, prehybridization was performed on the cDNA expression array using the ExpressHyb solution supplied with the kit. The probes were hybridized to the cDNA expression array in a Hybaid Micro 4 hybridization chamber (Hybaid, Teddington, UK) overnight at 68°C. Following hybridization, the arrays were washed and exposed to phosphor storage screens (Molecular Dynamics, Sunnyvale, CA, USA) overnight. The phosphor storage screens were viewed using IMAGEQUANT software (Molecular Dynamics, Sunnyvale, CA, USA) on a Phosphorimager. The images were analyzed for differential gene expression using ATLAS IMAGE 2.0 software (Clontech, Palo Alto, CA, USA) as previously described in detail from our laboratory.59 The arrays were stripped for subsequent hybridizations according to the manufacturer's protocol. Array analysis was carried out using Clontech's software as previously described in detail from our laboratory.59
RNA samples were treated for 30 min at 37°C with RNase-free DNase I (⩾5 g RNA) to remove contaminating DNA. The RNA sample quality was checked electrophoretically on a gel, and quantitatively by spectrophotometer analysis. Synthesis of cDNA was performed as detailed by Chong.59 PCR amplification was performed without reverse transcription in order to verify that there was no genomic DNA contamination prior to cDNA synthesis of the samples.
Two sets of primers were synthesized. The first set was designed to establish the identity of the GIP gene in the brain tissue, and the sequences were as follows: forward 5′-IndexTermGAGTTCCGATCCCATGCTAA-3′; reverse 5′-IndexTermCCCTCAGCACATCTTCATCA-3′. The second set was designed for real time RT-PCR. Sequences for real-time RT-PCR gene primers were obtained from the Stratagene database at www.stratagene.com. Primers with Tm (60°C) were synthesized by the Central Facility of the Institute for Molecular Biology and Biotechnology (MOBIX) at McMaster University.
Real-time RT-PCR for quantification of cDNA array results
Real-Time RT-PCR was performed in triplicate for each sample using MX 3000P real-time RT-PCR (Stratagene). The primers were: forward 5′-IndexTermGCTGAGGGACCTTCTGAT-3′; reverse 5′-IndexTermAGAGACTTTGGACCAGGG-3′. No primer-dimers were detected and transcripts showed optimal PCR efficiencies. An absolute standard curve was constructed with the use of corresponding pure amplicons from rat brain and rat small intestine as external standards in the range of 1 pg–10ag of cDNA. MX3000P real-time RT-PCR conditions were optimized to ensure that the amplifications were in the exponential phase, and that the efficiencies remained constant during the course of the PCR. Components of the reaction mixture included 100 ng of cDNA, 150 nM each (forward, reverse) of primers, 30 nM ROX (reference dye), 10 μl SYBR Green 2X Mix (Invitrogen), water to a final volume of 20 μl. All data were normalized against the cyclophilin housekeeping gene that was selected following the screening of several housekeeping genes. This gene's amplification remained unchanged between the two groups throughout the PCR reaction.
Isolation and sequencing of RT-PCR products
RT-PCR products and real-time RT-PCR products from striatal GIP and small intestine GIP were run on a 1.5% agarose gel with 0.5 μg/ml of ethidium bromide. The DNA bands were visualized under UV light and excised from the gel. The DNA was isolated using QIAquick Gel Extraction Kit (Qiagen, Mississauga, ON, Canada). The isolated bands were sequenced in both directions using GIP primers.
Radioimmunoassay for glucose-dependent insulinotropic polypeptide and estimation of insulin, triglycerides, glucose, and leptin
For the GIP assay, the peptide was extracted from the plasma using ethanol. Absolute ethanol was added to the plasma to give a final concentration of 70% (vol/vol). The samples were then mixed and centrifuged at 1600g for 20 min. The supernatant was transferred to a polypropylene tube and then lyophilized overnight and reconstituted in 250 μl of radioimmunoassay buffer supplied with the kit. The striatal tissue was sonicated in four volumes of acid–ethanol solution (82.5% ethanol and 1.9% hydrochloric acid); the extract was separated in the same way as the plasma samples above. The GIP radioimmunoassay kit was used according to the manufacturer's protocol (Peninsula Laboratories, San Carlos, CA, USA). According to kit manufacturer, the antibody displays 100% reactivity to porcine GIP and greater than 95% reactivity to rat GIP. Therefore, the GIP results reported here may have been underestimated by 5%.
Kits used for quantification of plasma concentrations of macromolecules were as follows: glucose (Johnson & Johnson, Clinical diagnostics, NY, USA), insulin and triglycerides (Sigma, Oakville, Onta, Canada), and leptin (Linco Research Incorporate, St Louis, MO, USA).
Immunohistochemistry for GIP localization within the striatum
Rat brain sections (20 μm) were cut and processed for immunolabeling of GIP as previously described from our laboratory.60 The images were captured under Zeiss LSM 510 confocal microscope. Double labeling with tyrosine hydroxylase was carried out using anti-tyrosine hydroxylase antibody (StressGen, Vancouver, BC, Canada).
cDNA array data, as well as real-time RT-PCR data (control versus clozapine treatment), were analyzed by Student's t-test. An expression change was considered significant at P<0.05.
- 5-HT :
- cAMP :
cyclic adenosine monophosphate
- CRE :
cAMP response element
- CREB :
cAMP response element-binding protein
- GIP :
glucose-dependent insulinotropic polypeptide
- GLUT4 :
- MMLV :
Moloney Murine Leukemia Virus
- PKA :
protein kinase A
- RT-PCR :
reverse transcriptase-polymerase chain reaction
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This work was supported by the Ontario Mental Health Foundation and National Institute of Health Grant NS20035 and the Canadian Institute of Health Research Grant MT2637.
Contacts: S Sondhi – firstname.lastname@example.org; JM Castellano – email@example.com; VZ Chong – firstname.lastname@example.org; RM Rogoza – email@example.com; KJ Skoblenick – firstname.lastname@example.org; BA Dyck – email@example.com; J Gabriele – firstname.lastname@example.org; N Thomas – email@example.com; ZB Pristupa – firstname.lastname@example.org; AN Singh – email@example.com; D MacCrimmon – firstname.lastname@example.org; P Voruganti – email@example.com; J Foster – firstname.lastname@example.org.
Preliminary results of this study were reported at the annual meeting of the Society for Neuroscience, 2003, San Diego CA, USA and at the Federation for American Societies of Experimental Biology Meeting in 2004, Washington, DC.
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Sondhi, S., Castellano, J., Chong, V. et al. cDNA array reveals increased expression of glucose-dependent insulinotropic polypeptide following chronic clozapine treatment: role in atypical antipsychotic drug-induced adverse metabolic effects. Pharmacogenomics J 6, 131–140 (2006) doi:10.1038/sj.tpj.6500346
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