Curcumin Inhibits Adipogenesis Elicited by Clozapine in 3 T 3L 1 Cells

Although clozapine (CZP), which is used for schizophrenia treatment, causes weight gain, the mechanism remains unclear. We recently reported that the naturally occurring compound curcumin (CUR) suppresses adipogenesis in 3T3-L1 cells. The aims of the present study were to determine the mechanism by which CZP induces adipocyte differentiation of 3T3-L1 cells, and whether CUR reduces CZP-induced adipogenesis. We found that cells grown in the presence of CZP had significantly higher triacylglycerol levels, numbers of lipid-filled adipocytes, and mRNA expression levels of CCAAT-enhancer binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ) than those grown without CZP. Treatment with CZP plus CUR resulted in major reductions in these four parameters. These results suggest that CZP enhances adipogenesis in 3T3-L1 cells via the C/EBPα-PPARγ pathway and that by interrupting CZP’s effects, CUR might be a potent agent for preventing CZP-induced weight gain.


Introduction
Clozapine (CZP) is a second-generation atypical antipsychotic drug used to treat many schizophrenia symptoms, such as positive symptoms, negative symptoms, and recognition disorders.The major side effects of CZP are weight gain and diabetes mellitus, which are risk factors for cardiovascular diseases [1].The probable causes of the metabolic adverse effects are poor satiety and increased food intake due to blockade of receptors such as 5-hydroxytryptamine 2C S. Sakuma et al. (5HT 2C ), dopamine D2 (D 2 ), and histamine 1 (H 1 ) in the central nervous system [2].In the last decade, it has been suggested that second-generation atypical antipsychotic drugs cause body weight gain not only by affecting the receptors of the central nervous system but also by directly affecting peripheral adipogenesis [3]- [8].However, the mechanism by which second-generation atypical antipsychotic drugs directly induce adipocyte differentiation remains unclear.
Over the years, many phytochemical compounds have been found to have a high capacity to act against various disorders, including metabolic syndrome.
One of these substances, curcumin (CUR), a nutraceutical and pharmaceutical, is the active ingredient in turmeric, a well-known Indian spice derived from the dried roots of the Curcuma longa plant.The effects of CUR in metabolic syndrome have been studied in several clinical trials.In these trials, CUR improved the lipid profiles and modified the cholesterol-related parameters of the subjects [9] [10].Furthermore, CUR can improve anthropometric measurements and body composition when taken as part of the diet together with the implementation of appropriate lifestyle interventions [11].

Materials
Mouse 3T3-L1 preadipocytes were obtained from the European Collection of Cell Cultures (Wiltshire, UK).Transcriptor First Strand cDNA Synthesis kit and Light Cycler First Start DNA Master plus SYBR green reagent were obtained from Roche Diagnostics (Indianapolis, IN, USA).TRIzol reagent and the primers for β-actin, PPARγ, and C/EBPα were purchased from Invitrogen (Carlsbad, CA, USA).CZP, CUR, olanzapine (OZP), chlorpromazine (CPZ) and a protease inhibitor cocktail were obtained from Sigma Chemical Co.(St.Louis, MO, USA).
The triglyceride E-test Wako kit was obtained from Wako Pure Chemical Industries, Limited (Osaka, Japan).Lipid Droplets Fluorescence Assay kit was obtained from Cayman Chemical Co.(Ann Arbor, MI, U.S.A.).All other reagents used were of analytical grade.

Treatments with CZP and CUR
CZP and CUR were prepared in dimethyl sulfoxide (Me 2 SO) and added to the growth, initiation, and maturation media from day 3 (time of addition of dexamethasone, IBMX, and insulin) to day 9 (the end of the experiment).The Me 2 SO concentration was maintained up to 0.25% of the total volume of the medium.Preliminary experiments demonstrated no significant effects of 0.25% v/v Me 2 SO on cell differentiation.

Oil Red O Staining
The cells were fixed in 4% formaldehyde-phosphate buffer (pH 7.4) for 1 h, rinsed with water, and stained with 0.3% Oil Red O dye for 1 h.After washing again with water, the cells were monitored using a microscope (10 × 20 magnification).

Quantification of Mature Adipocytes
Mature adipocytes in the culture were quantified using Lipid Droplets Fluores- were collected.The results are expressed as the percentage of cells in the P2 region.

Determination of TG Levels
The cells were harvested by scraping them from the culture dishes into lysis buffer (1% Triton-100, 150 mM NaCl, 4 mM ethylenediaminetetraacetic acid, and 20 mM Tris-HCl [pH 7.4] containing a protease inhibitor cocktail) and lysed completely using a horn-type sonicator.The TG level, which is an index of lipid accumulation, was determined using Triglyceride E-test Wako kit following normalization of the protein amounts and expressed as TG content (µg/mg protein).

Statistical Analysis
The results are expressed as the mean ± standard error of the mean values.Significant differences in data between two groups were assessed using Student's t-test.Differences were considered statistically significant at P < 0.05.CZP, but not OZP, promoted TG accumulation with statistical significance.The findings show that second-generation atypical antipsychotic drugs cause body weight gain partially by affecting peripheral adipogenesis directly.This result is in agreement with results of previous studies [3]- [8].

CUR Interferes with CZP-Induced Adipogenesis in 3T3-L1 Cells
To study the potential inhibitory effects of CUR on CZP-induced adipogenesis, 20 µM CUR was added to the culture medium together with CZP (20 µM) during the differentiation of the 3T3-L1 cells.Changes in differentiation were monitored by determining the TG levels (Figure 1

CUR Disrupts the Effect of CZP at the Site of the C/EBPα-PPARγ Pathway
As shown in Figure 3

Discussion
Generally, patients responding to CZP should continue maintenance treatment on their effective dose beyond the acute episode.Several studies have assessed the relationship between plasma CZP levels and clinical responses [21].Most studies have found that a threshold plasma level of Together with C/EBPα, PPARγ promotes terminal differentiation by activating the expression of downstream adipocyte-specific genes.
In the present results, we showed that CZP at 5 µM or less concentration potentiated TG accumulation in 3T3-L1 cells (Figure 1(a)).Additionally, compared to exposure to DM alone, 20 µM CZP exposure significantly increased the mRNA expression levels of PPARγ and C/EBPα (Figure 3).Thus, the present study confirms that CZP at clinical levels causes body weight gain partially by directly affecting peripheral adipogenesis.It might be also possible that the CZP-induced adipogenesis is partially ascribed to enhancement of the C/EBPα-PPARγ pathway.
Humans can consume up to 8 grams of CUR per day without experiencing toxic effects [24]; this makes CUR a very interesting potential preventive agent against various diseases.CUR has been reported to inhibit adipogenesis in both cellular and animal models of obesity by several research groups, including ours [9]- [15].In addition, recent clinical trials have demonstrated that CUR can reduce blood TG levels in patients with type II diabetes [25] [26].
In the present study, the level of adipogenesis in cells treated with 20 µM CZP plus 20 µM CUR was almost identical to that in the cells that were not treated with CZP (Figure 1(b) and Figure 2).Furthermore, the results shown in Figure 3 demonstrate that CUR reversed the CZP-induced increments in the mRNA expression of PPARγ, and C/EBPα.These results suggest that because of its ability to inhibit the effects of CZP, CUR might be a potent naturally occurring compound that prevents CZP-induced weight gain, thus preventing obesity and worsening of obesity-related complications.

Conclusion
In conclusion, to our knowledge, this is the first study to show that CUR could inhibit CZP-induced weight gain and its complications via inhibition of adipocyte differentiation.Our results suggest that CUR has the potential to prevent the side effects of atypical antipsychotic drugs.
Assay kit, and flow cytometry.The cells were trypsinized carefully and centrifuged at 4˚C and 200 ×g for 5 min.The cell pellet was resuspended, fixed, and stained with the lipophilic fluorescent dye Nile Red according to the protocol received with Lipid Droplets Fluorescence Assay kit.The samples were then analyzed with a FACSAria III flow cytometer (Becton Dickinson, Basel, Switzerland).Nile Red fluorescence was measured on the FL2 emission channel through a 585 ± 21 nm bandpass filter following excitation with an argon ion laser source at 488 nm.Using a forward scatter (FSC)/side scatter (SSC) representation of events, the P1 region was defined to exclude cellular debris from the analysis.A selection window called P2 was then established on the FL2/FSC blot of the P1 population to count cells with high FL2 values (mature adipocytes) as described by Sottile and Seuwen [20].Data analysis was performed using BD FACSDiva Software ver.8.0 (Becton Dickinson).For each sample, 20,000 events DOI: 10.4236/fns.2018.95044586 Food and Nutrition Sciences S. Sakuma et al.
Levels of β-Actin, PPARγ, and C/EBPαUntreated cells, as well as those treated with CZP or CZP plus CUR up to day 5, were washed with ice-cold phosphate-bufferedsaline.Total cellular RNA was prepared using TRIzol reagent.One microgram of total RNA was reverse-transcribed into cDNA by using the Transcriptor First Strand cDNASynthesis kit.Next, the concentration and quality of the purified total RNA were determined spectrophotometrically at 260 nm and by the OD 260:280 ratio.mRNA expression was determined by real-time reverse transcription polymerase chain reaction using Light Cycler First Start DNA Master plus SYBR Green reagent and a Light Cycler instrument.The results are expressed as the target mRNA level relative to that of β-actin mRNA, and the values obtained in the presence or absence of the drugs were expressed relative to the values associated with exposure to the differentiation medium (DM) alone.The primers used for β-actin, PPARγ, and C/EBPα were as follows: β-actin, 5'-ACACCCCAGCCATGTACG-3', 5'-TGGTGGTGAAGCTGTAGCC-3'; PPARγ, 5'-GTGAAGCCCATCGAGGACA-3', 5'-TGGAGCACCTTGGCGAACA-3'; and C/EBPα, 5'-ATGGTTTCGGGTCGCTGGAT-3', 5'-CCACGGCCTGACTCCCTCAT-3'.

Figure 1 (Figure 1 .
Figure 1(a) shows the effect of CZP, OZP and CPZ at concentrations ranging from 1 to 20 µM on TG level during the differentiation of 3T3-L1 preadipocytes into mature adipocytes.Exposure to the second-generation atypical antipsychotics CZP and OZP increased TG levels, whereas exposure to CPZ, a classical neuroleptic drug that primarily acts on dopamine D 2 receptors, did not.At 5 µM, (b)), Oil Red O staining (Figure 2(a)), and determining the percentage of mature adipocytes (Figure 2(b) and Figure 2(c)).Compared to exposure to DM alone, exposure to both 20 µM CZP and DM resulted in a significant increase in TG levels (1.3 fold in Figure 1(b)), Oil Red O-stained cells (Figure 2(a)), and percentage of lipid-filled adipocytes as shown by Nile Red and flow cytometry (2.4 fold in Figure 2(c)).However, the administration of 20 µM CUR with CZP almost nullified the effects of CZP; the TG levels (Figure 1(b)), number of Oil Red O-stained cells (Figure 2(a)), and percentage of lipid-rich adipocytes (Figure 2(c)) were similar to the respective levels in the presence of DM or CUR alone.The results shown in Figure 1(b) and Figure 2 demonstrate that CUR interferes with the CZP-induced adipogenesis in 3T3-L1 cells.

Figure 2 .Figure 3 .
Figure 2. Alterations in results of (a) Oil Red O staining and (b) Nile red staining with flow cytometry, and (c) percentages of cells stained by Nile red (P2), in 3T3-L1 adipocytes treated with clozapine (CZP) and curcumin (CUR).(a) The photographs shown are representative of results from six independent experiments.The cells stained by Oil Red O were visually monitored by microscopic observation (10 × 20 magnification).CZP, 20 µM; CUR, 20 µM; (b) A representative flow cytometric analysis using Nile red staining; (c) Data are presented as mean ± S.E.values (n = 4).α indicates P < 0.01 compared to the untreated cells (None).β indicates P < 0.01 compared to the cells cultured in the differentiation medium (DM).γ indicates P < 0.01 compared to the cells treated with CZP alone.CZP, 20 µM; CUR, 20 µM.
[1] -2.6 µM CZP is associated with an increased probability of a good clinical response to the drug.During continuous dosage of CZP, weight gain is a common side effect of this drug[1].Short-term weight gain leads to poor adherence and other deleterious effects.However, the mechanism underlying CZP-induced weight gain is not DOI: 10.4236/fns.2018.95044590 Food and Nutrition Sciences S. Sakuma et al.