Elsevier

Brain Research

Volume 923, Issues 1–2, 27 December 2001, Pages 82-90
Brain Research

Research report
Inhibition of glucose transport in PC12 cells by the atypical antipsychotic drugs risperidone and clozapine, and structural analogs of clozapine

https://doi.org/10.1016/S0006-8993(01)03026-8Get rights and content

Abstract

Treatment of schizophrenics with some antipsychotic drugs has been associated with an increased incidence of hyperglycemia and new-onset type 2 diabetes. Some of these drugs also inhibit glucose transport in rat pheochromocytoma (PC12) cells. The current study was designed to examine the effects of the atypical antipsychotic drugs — risperidone, clozapine and analogs of clozapine on glucose uptake in PC12 cells. Glucose transport was measured in cells incubated with vehicle or drug over a range of concentrations (0.2–100 μM). Uptake of 3H-2-deoxyglucose was measured over 5 min and the data were normalized on the basis of total cell protein. Risperidone and clozapine inhibited glucose transport in a dose-dependent fashion with IC50’s estimated to be 35 and 20 μM, respectively. The clozapine metabolite, desmethylclozapine, was considerably more potent than the parent drug, whereas clozapine N-oxide was essentially inactive. The structural analogs of clozapine, loxapine and amoxapine, both inhibited glucose transport with amoxapine being the least potent. The ability of the drugs to inhibit glucose transport was significantly decreased by including 2-deoxyglucose (5 mM) in the uptake medium. Schild analysis of the glucose sensitivity of clozapine, loxapine and risperidone indicated that 2-deoxyglucose non-competitively antagonized the inhibitory effects of these drugs. Moreover, clozapine and fluphenazine inhibited glucose transport in the rat muscle cell line, L6. These studies suggest that the drugs may block glucose accumulation directly at the level of the glucose transporter (GLUT) protein in cells derived from both peripheral and brain tissue. Furthermore, this work may provide clues about how the antipsychotic drugs produce hyperglycemia in vivo.

Introduction

Glucose is transported into cells by a family of facilitative, sodium-independent glucose transport (GLUTs) proteins, primarily GLUT3 in neurons [28] and GLUT1 and GLUT4 in muscle cells [23]. Recently, various antipsychotic drugs have been shown to inhibit this process in neuronal cells [12], [14]. Moreover, the same drugs can affect glucose metabolism in man leading to hyperglycemia and even diabetes in some patients [3], [20], [32], [40]. The atypical antipsychotic drug clozapine, is a useful starting point for the examination of the hyperglycemic response because it blocks glucose accumulation in cells [12], [14] and because of the availability of major metabolites and drugs with closely related structures.

Clozapine is thought to exert its antipsychotic effects primarily through the antagonism of dopamine and serotonin receptors in brain (reviewed in Ref. [37].) Because clozapine can induce fatal agranulocytosis [24], it is mainly used in patients who do not respond to other antipsychotic drugs. Other serious side effects that have been reported for clozapine include significant weight gain, hyperglycemia and diabetes [22], [26], [34], [19]. The causes of these various metabolic disturbances have not been determined. Insulin responsive tissues, such as muscle, are important for maintaining proper blood glucose concentrations [10]. These peripheral tissues could be affected by the antipsychotic drugs, which might contribute to the development of hyperglycemia and diabetes. In addition, muscle cells express GLUT isoforms, primarily GLUT1 and GLUT4 [23] that are distinct from those found in neurons (primarily GLUT3). Therefore, we have examined the effect of clozapine and fluphenazine on a representative peripheral tissue, specifically the rat L6 muscle cell line.

Clozapine is extensively metabolized in the liver to clozapine N-oxide and desmethylclozapine [16]. It is not known whether these metabolites, like the parent drug, inhibit glucose uptake and accumulation in cells. Clozapine and desmethylclozapine are found at significant concentrations in plasma and brain tissue [6]. Clozapine and desmethylclozapine significantly antagonize dopamine and serotonin receptor systems in vitro [25] and there is evidence that both compounds similarly affect the CNS of rats [45]. On the other hand, serum concentrations of clozapine N-oxide are low [6] and this metabolite is much less potent than clozapine and desmethylclozapine in receptor binding assays [25]. It is not known whether the metabolites of clozapine would affect glucose transport to the same extent as the parent drug. Therefore, studies of clozapine and its major metabolites were conducted in an attempt to define a structure–activity relationship (SAR) among a group of compounds that are closely related.

Through this research it may be possible to clarify the relationship between the structure of an antipsychotic drug and its ability to inhibit glucose uptake. Loxapine, an atypical antipsychotic, and amoxapine, an antidepressant, are both structurally related to clozapine and both have been reported to cause hyperglycemia in man [41]. On the other hand, the older psychotropic drugs (phenothiazines), that inhibit glucose transport in cells and cause diabetes, are structurally distinct from clozapine [12], [14]. Risperidone, another atypical antipsychotic drug, is also structurally different from clozapine, although it shares some of clozapine’s side effects [44]. Thus, a diverse panel of psychotropic drugs is available for these studies, including drugs that closely resemble clozapine (e.g. desmethylclozapine and loxapine) in terms of their structure and their adverse side effects.

The mechanisms through which the antipsychotic drugs affect glucose accumulation in PC12 cells are not known. Although these cells bear dopamine receptors [35], the failure of the D2 dopamine receptor antagonists, haloperidol and sulpiride, to inhibit glucose uptake suggests that the effects of the antipsychotic drugs on transport are not mediated by this receptor [12]. Direct interaction of the drugs with GLUTs is one possible mechanism for inhibiting glucose transport. Cytochalasin B binds directly to an intracellular portion of the GLUT protein and inhibits transport in a glucose-sensitive manner [21]. It is not known whether the psychotropic drugs that inhibit glucose uptake are, like cytochalasin B, sensitive to glucose concentration. One goal of these studies was to determine if the clozapine-related drugs and risperidone inhibit the uptake of glucose in a glucose-sensitive manner.

Section snippets

Reagents and drugs

Clozapine, clozapine N-oxide, N-desmethylclozapine, loxapine, amoxapine and risperidone were purchased from Research Biochemicals International (Natick, MA). Poly-l-lysine (PLL) and 2-deoxyglucose (2-DOG) were obtained from Sigma Chemical Company (St. Louis, MO).

Cell lines

PC12 cells were obtained from American Type Culture Collection (ATCC) and were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% equine serum, 5% fetal bovine serum, and 2% penicillin/streptomycin as previously

Chemical structures

Fig. 1 shows the chemical structures of the drugs used in this study. Clozapine with its two major metabolites, desmethylclozapine and clozapine N-oxide, are shown on the top row. The antipsychotic loxapine and the tricyclic antidepressant amoxapine are both structural analogs of clozapine. Risperidone is an atypical antipsychotic that is structurally distinct from clozapine.

Concentration–response curves

Previous studies have shown that several antipsychotic drugs, including clozapine, can inhibit glucose transport in

Discussion

Previously, it has been reported that chlorpromazine, fluphenazine, and clozapine inhibited glucose transport in PC12 cells [14]. The current work expands the list of psychotropic agents that inhibit glucose uptake in neuronal cells to include desmethylclozapine, loxapine, amoxapine, and risperidone. In addition, this is the first demonstration of the ability of the antipsychotic drugs clozapine and fluphenazine to inhibit glucose transport in a cell type that was not of neuronal origin — the

Acknowledgements

The authors would like to thank Ye Liu for assistance in performing this work. This study was supported by the Department of Psychiatry, LSUHSC-Shreveport.

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