Introduction

Second-generation antipsychotics (SGA) are the most common drugs used for treatment of schizophrenia and have a reduced risk of extrapyramidal symptoms compared to first-generation antipsychotics. However, most SGA are linked to weight gain, impaired glucose tolerance and lipid abnormalities, resulting in the development of various metabolic disorders. A high prevalence of metabolic syndrome (McEvoy et al., 2005; Birkenaes et al., 2007) and an increased mortality rate (Laursen et al., 2007) were observed in schizophrenic patients. The risk of weight gain is variable among SGA. Olanzapine and clozapine are associated with the highest risk of weight gain, followed by chloropromzaine, resperidone, and quetiapine (Lett et al., 2012). Ziprasidone belongs to the group with the lowest risk of weight gain, and it has even shown to reduce weight gain by increasing resting energy expenditure without decreasing food intake (Park et al., 2012). The rank order of SGA for inducing weight gain is consistent with that of their metabolic side effects (Newcomer, 2005, 2007), supporting the idea that drug's risk for adverse metabolic changes are strongly associated with its potency for increasing adiposity. Although differential binding affinity of individual SGA to histamine, serotonin, α-adrenergic receptors (Kroeze et al., 2003; Matsui-Sakata et al., 2005; Deng et al., 2010) or PKC-β activation (Pavan et al., 2010) has been suggested as possible mechanisms, the biochemical and pharmacological bases of SGA-induced weight gain are mostly unclear.

Brown adipose tissue (BAT) serves a specialized function in adaptive thermogenesis and plays an opposite role to that of white adipose tissue by burning metabolic substrates instead of storing energy in the form of triglycerides. Therefore, BAT has long been accepted as an important metabolic organ in small mammals such as mice or rats, but was previously considered to have little physiological relevance in humans beyond early childhood. However, several lines of evidence using 18F-fluorodeoxyglucose positron emission tomography clearly confirmed the existence of active BAT in humans (Nedergaard et al., 2007; Lichtenbelt et al., 2009). Moreover, recent studies also suggested a possible role of BAT in human energy metabolism. BAT activation is either stimulated by cold, insulin, and catecholamines (Orava et al., 2011; Wang et al., 2011) or down-regulated by central obesity and bariatric surgery (Wang et al., 2011; Vijgen et al., 2012). Promoting brown adipocyte differentiation leads to an increase in energy expenditure and a reduction in weight gain (Tseng et al., 2008), whereas reduced brown adipocyte differentiation is associated with obesity and insulin resistance in humans (Yang et al., 2003). To the best of our knowledge, only two reports have suggested a possible involvement of BAT in psychotropic-induced obesity. Lithium, which is primary therapy for bipolar disorder and known to induce obesity in humans, inhibited the differentiation of mouse brown adipocytes (Rodríguez de la Concepción et al., 2005). In the sole physiological study, clozapine reversed the thermogensis of brown adipose tissue in rabbits (Blessing et al., 2006). Taken together, these findings suggest that SGA are very likely to induce weight gain by the inhibition of brown adepogenesis. Therefore, we examined the relevance between SGA and weight gain by comparing the effect of clozapine, quetiapine, and ziprasidone on the differentiation of mouse brown preadipocytes. In this study, we provide for the first time biochemical and molecular biological evidence showing that SGA may induce weight gain and metabolic disturbances by inhibiting brown adipocyte differentiation.

Results and Discussion

Rationale for the selection of drugs and their concentration

To reveal the possible involvement of BAT in clozapine- or quetiapine-induced weight gain, we examined the effect of clozapine, quetiapine, and ziprasidone on the brown adipocyte differentiation. For a more rational and objective comparison, we selected the concentration of each drug at which they exert similar clinical efficacies based on the recent therapeutic reference ranges (TRR) recommended by the Arbeitsgemeinschaft fur Neuropshychopharmakologie und Pharmakopsychiatrie (AGNP) (Hiemke et al., 2011). In addition, since a previous report showed that clozapine did not increase intracellular triglyceride content during adipogenic differentiation of human adipose-derived stem cells at 30 µM, which was 16-fold higher than the upper limit concentration of TRR (Sertié et al., 2011), we evaluated the effect of the drugs at the concentrations that were approximately 20-fold higher than the upper limit of TRR, i.e. clozapine (40 µM), quetiapine (30 µM), and ziprasidone (10 µM).

Effects of drugs on the adipogenic differentiation of brown preadipocytes

To disclose the involvement of BAT in three SGA-induced weight gains, the effect of the drugs on the differentiation of the brown preadipocyte cell line was examined. We induced adipogenic differentiation for 8 days in the presence of clozapine (40 µM), quetiapine (30 µM), and ziprasidone (10 µM), and examined intracellular accumulation of triglyceride by Oil Red-O staining. As shown in Figure 1, brown adipocyte differentiation was suppressed almost completely and partly by clozapine and quetiapine, respectively. However, ziprasidone did not show any effect on the differentiation of brown preadipocytes. When we examined the effect on cell viability, clozapine (1.25-40 µM), quetiapine (10-60 µM), and ziprasidone (2-12 µM) did not demonstrate any cytotoxicity during the adipogenic differentiation period (Supplemental Data Figure S1). These results imply that the inhibitory potency of individual drug on brown adipogenesis may be proportionate to its risk for the induction of weight gain and metabolic disturbances. It is noteworthy that clozapine inhibited the differentiation of brown preadipocytes in our results while it did not induce any changes in white preadipocytes (Hu et al., 2010; Sertié et al., 2011). In addition, olanzapine and resperidone, which showed a similar weight gain risk to clozapine and quetiapine, respectively, also induced adipogenesis in 3T3-L1 cells (Yang et al., 2007; Hu et al., 2010). These findings strongly suggest the differential regulatory role of SGA in the differentiation of white and brown preadipocytes. Ziprasidone (0.2 µM) and clozapine (40 µM) were shown to be highly toxic during adipogenic differentiation from adipose-derived stem cells (Sertié et al., 2011), but in our results, the viability of brown preadipocytes was absolutely not affected even at a much higher (ziprasidone, 10 µM) or at the same (clozapine, 40 µM) concentrations (Supplemental Data Figure S1). Although there are significant differences between these two cell types, these findings suggest the possibility that brown preadipocytes may be more resistant to the cytotoxic activity of SGA than white preadipocytes. In accordance with this point of view, Nisoli et al. (2006) reported that white adipocytes are less prone to apoptotic stimuli than brown adipocytes. Further experiments are needed to investigate the differential responses of white and brown (pre)adipocytes to SGA.

Figure 1
figure 1

Effect of clozapine, quetiapine and ziprasidone on brown adipocyte differentiation. Brown preadipocytes were differentiated with the induction media in the presence of clozapine (40 µM), quetiapine (30 µM), and ziprasidone (10 µM) or control (0.1% DMSO). At day 8, fully differentiated cells were stained with Oil Red-O and photographed by phase-contrast microscopy. Representative data from three independent experiments are shown (Original magnification, 100 ×).

Full size image

Effects of drugs on the expression of brown adipocyte markers

Despite the difference in the developmental origin and physiological function of brown and white adipocytes, both cell types share a very similar transcriptional cascade. Indeed, PPARγ is absolutely necessary for both white and brown fat development (Kajimura et al., 2010). Moreover, C/EBPβ induces PGC-1α (Wang et al., 2008), and has been recently suggested as a critical transcription factor for the initiation of brown fat formation (Kajimura et al., 2009). However, the development of the specific characteristics of brown adipocytes requires the expression of additional transcriptional regulators. PRDM16, a zinc finger protein, activates PPARγ2, forms a transcriptional complex with C/EBPβ (Kajimura et al., 2009), and finally enhances brown adipogenesis by the induction of brown fat-selective markers such as PGC-1α, UCP-1, and Cidea (Kajimura et al., 2009). To provide molecular evidence for our results showing SGA-induced inhibition of brown adipogenesis (Figure 1), we examined the effect of SGA on the mRNA expression of PRDM16, C/EBPβ, PPARγ2, UCP-1, PGC-1α, and Cidea at 8 days after the induction (Figure 2) or during the differentiation of brown preadipocytes (Figure 3, Supplemental Data Figure S2). The results revealed that clozapine significantly inhibited the expression of all genes tested (P < 0.05), and particularly PRDM16, UCP-1 and Cidea were almost disappeared by day 8 (P < 0.05) (Figure 2A). Inhibitory effect of clozapine was time-dependent, and this was apparent from day 4 for PRDM16 and PPARγ2 (Figure 3). Quetiapine also down-regulated the expression of PRDM16, PPARγ2, UCP-1, and Cidea at day 8 after the induction (P < 0.05), but the intensity was much weaker than clozapine (Figure 2B). Inhibitory effect of quetiapine was also not consistent as that of clozapine, for example, CEBPβ was transiently inhibited only at days 4 and 6 (Figure 2C, Supplemental Data Figure S2A). In contrast to clozapine and quetiapine, ziprasidone showed inhibitory activity only in the early stage of the differentiation, and PPARγ2 expression was significantly increased at days 6 and 8 (P < 0.05) (Supplemental Data Figure S2B). These findings clearly correspond with the results from Oil Red-O staining (Figure 1), and strongly support our hypothesis that the inhibition of brown adipogenesis may be one of the mechanisms for SGA-induced weight gain. Among the genes tested in our stuy, transcriptional regulation of PRDM16 by clozapine and quetiapine is remarkable since they are considered as master regulators for brown adipogenesis. Brown adipogenesis from white preadipocytes or myoblasts was successfully induced by the ectopic expression of PRDM16 (Seale et al., 2007), and BAT from PRDM16-/- mice exhibited an abnormal morphology and reduced expression of brown fat markers (Seale et al., 2008). Recently, association of PRDM16 with metabolic syndrome was also suggested (Statistical analysis

All values are expressed as the means ± SEM from at least three independent experiments. All statistical calculations were performed using GraphPad Prism 5.03 software (GraphPad Software Inc., San Diego, CA). One- or two-way ANOVA followed by Turkey's Multiple Comparison Test was used to compare means. A probability (P) value less than 0.05 was considered to be significant.