Anti-adipogenic effect of a turmeric extract-loaded nanoemulsion in 3T3-L1 preadipocytes and high fat diet-fed mice

An increase in the number of adipocytes (hyperplasia) due to adipocyte differentiation and an increase in the size of adipocytes (hypertrophy) are both contributors to the development of obesity (Guo et al., 2015; Shepherd et al., 1993; Spalding et al., 2008). An increase in adipose tissue mass is paralleled by higher prevalence of obesity-related complications, such as hyperlipidemia, hypertension, type 2 diabetes, and nonalcoholic fatty liver disease (Kopelman, 2000; Spalding et al., 2008). An improved understanding of adipocyte differentiation and function is critical to accelerate the development of drugs for the treatment of obesity and its complications.


Introduction
An increase in the number of adipocytes (hyperplasia) due to adipocyte differentiation and an increase in the size of adipocytes (hypertrophy) are both contributors to the development of obesity (Guo et al., 2015;Shepherd et al., 1993;Spalding et al., 2008). An increase in adipose tissue mass is paralleled by higher prevalence of obesity-related complications, such as hyperlipidemia, hypertension, type 2 diabetes, and nonalcoholic fatty liver disease (Kopelman, 2000;Spalding et al., 2008). An improved understanding of adipocyte differentiation and function is critical to accelerate the development of drugs for the treatment of obesity and its complications.
The cellular and molecular mechanisms underlying adipocyte differentiation have mainly been established by studying in vitro models of preadipocytes derived from the mouse, such as the 3T3-L1 and 3T3-F442A cell lines (Gregoire et al., 1998;Rosen & MacDougald, 2006). Upon treatment with inducers of adipogenesis, including glucocorticoid, insulin, and inhibitors of cyclic AMP phosphodiesterase, a process known as mitotic clonal expansion is initiated, causing growth-arrested confluent preadipocytes to re-enter the cell cycle, thereby contributing to adipocyte hyperplasia (Haslam & James, 2005;Rosen & MacDougald, 2006). Adipogenesis is mediated by the activation of a programmed series of gene transcription events, which involves the upregulation of adipogenic transcription factors, such as CCAAT/enhancer-binding protein (C/EBP) α/β and peroxisome proliferator-activated receptor (PPAR) γ. This leads to higher expression of adipocyte protein 2 (aP2), a marker of terminal adipocyte differentiation (Coe et al., 1999;Farmer, 2006;Guo et al., 2012;Rosen & MacDougald, 2006;Wakabayashi et al., 2009).
Turmeric, Curcuma longa L., a perennial herb of the Zingiberaceae family, has been widely used for centuries as a spice, food preservative, coloring material, and traditional medicine in South Asia, Africa, and Latin America, including in Ondia and China (Araújo & Leon, 2001;Gupta et al., 2013). Although the major compound responsible for the biological activities of turmeric is curcumin, which constitutes 2-5% of the raw plant material, numerous other chemical entities, including alkaloids, sterols, polyphenols, and terpenoids, have been identified in turmeric and these also exhibit diverse pharmacological effects (Aggarwal et al., 2013;Gupta et al., 2013). These effects include amelioration of carrageenan-induced inflammation (Mukhopadhyay et al., 1982) and lipid peroxidation (Reddy & Lokesh, 1992;Reddy & Lokesh, 1994), anti-tumor effects (Huang et al., 1988;Huang et al., 1991), and anti-bacterial (Sathishkumar et al., 2010;Yano et al., 2006) and anti-protozoal activities (Rasmussen et al., 2000). On addition, the ethyl acetate fraction of turmeric partially inhibits lipogenesis by regulating expression of genes involved in glucose uptake and lipolysis (Lee et al., 2010). Furthermore, turmeric polyphenols protected adipocytes against oxidative stress by inhibiting the hydrogen peroxide-induced production of proinflammatory cytokines and reactive oxygen species via Anti-adipogenic effect of a turmeric extract-loaded nanoemulsion in 3T3-L1 preadipocytes and high fat diet-fed mice upregulation of antioxidant enzymes, such as superoxide dismutase and catalase (Septembre-Malaterre et al., 2016). However, the cellular and molecular mechanisms underlying the biological effects of turmeric extracts on adipocyte differentiation have not been fully elucidated.
On this study, we have investigated the effects of a turmeric extract-loaded nanoemulsion (TENE) on adipogenesis in 3T3-L1 adipocytes and in mice fed a high fat diet (HFD), paying particular attention to the expression of the genes implicated in adipocyte differentiation (PPARγ, C/EBPα, and aP2). Dur results indicate that TENE inhibits adipogenesis by reducing expression of adipogenic transcription factors, and therefore has the potential to ameliorate metabolic disorders related to obesity.

Preparation and characterization of Turmeric Extract-Loaded Nanoemulsion (TENE)
Turmeric powder was extracted using 50% ethanol and shaking for 24 h, and the solvent was evaporated under vacuum to concentrate the extract. TENE was prepared by ultrasonication as follows. The oil phase was prepared by dissolving 173.92 g turmeric extract and 86.96 g MCT oil containing 26.09 g soy lecithin, while the aqueous phase was prepared by mixing 17.39 g tween-80, 173.9 mL turmeric extract, and 869.57 mL distilled water. A coarse emulsion was prepared by stirring at room temperature for 2 h. Then, a nanoemulsion was prepared by high speed homogenization (HG-15D, Daihan Scientific Co., Ltd., Wonju, Korea) of the coarse emulsion at 5,000 rpm for 10 min, with ultrasonication using a Vibra Cell (VCX-750, Sonics & Materials, Onc., Sandy Hook, USA) for 15 min, and a high pressure homogenization under 10,000 psi for three cycles. TENE powder was prepared by spray drying (KL-8, Seo Gang Engineering Co., Ltd., Cheonan, Korea) of 1,000 mL TENE with 113.06 g dextrin. The curcumin content of the TENE was determined by spectrophotometric methods. To determine the physicochemical properties of the TENE, polydispersity index (PDO), zeta potential, and the mean droplet size of TENE were determined using a zeta potential and particle size analyzer (ELSZ-1000, Dtsuka Electronics Co., Ltd., Dsaka, Japan), with a detector angle of 90° and a wavelength of 633 nm at 25 °C.

Culture and differentiation of 3T3-L1 preadipocytes
HepG2 cells and 3T3-L1 preadipocytes obtained from the Korean Cell Line Bank (Seoul, Korea) were maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (Welgene, Daegu, Korea) or 10% newborn calf serum (Thermo Fisher Scientific, MA, USA), respectively, in the presence of 100 U/mL penicillin and 100 μg/mL streptomycin, in a humidified atmosphere of 5% CD 2 at 37 °C. To differentiate 3T3-L1 preadipocytes into adipocytes, confluent 3T3-L1 cells maintained in DMEM containing 10% fetal bovine serum (Welgene) were differentiated into adipocytes (starting on day 0) using a mixture of 1 μg/mL insulin, 1 μM dexamethasone, and 0.5 mM OBMX (MDO). The culture medium was then replaced with complete DMEM containing 1 μg/mL insulin, and the cells were incubated for another 4 days. Following this, the cells were incubated in complete DMEM, replacing the medium every other day. After incubation for 8 days, the extent of adipogenic differentiation was assessed.

Determination of cell viability
Cell viability was determined by the trypan blue exclusion method and MTT assay. For trypan blue exclusion, 3T3-L1 cells were seeded in 24-well culture plates at a density of 1 × 10 4 cells per well. Following incubation for 24 h, the cells were treated with the indicated concentrations of TENE for 24 h. They were then washed with ice-cold phosphate-buffered saline (PBS) and harvested. After adding 0.4% trypan blue solution, the number of viable cells in the suspension was counted with a hemocytometer. MTT assay was performed 3T3-L1 cells seeded in 48-well culture plates at a density of 3 × 10 3 cells per well. The cells were incubated with various concentrations of TENE for 24 h, and then MTT solution was added to the culture medium. After incubation for 4 h, the medium was removed, the crystal formazan was dissolved, and the absorbance at 570 nm was determined.

Analysis of lipid content by Oil red O staining
3T3-L1 cells differentiated with MDO in the presence or absence of TENE were washed twice with ice-cold PBS and then fixed in 4% paraformaldehyde for 10 min at room temperature. After washing with PBS, the cells were stained with Dil Red D solution (6 parts 0.5% Dil Red D dye in isopropanol and 4 parts water) for 2 h at room temperature. Following extensive washing with distilled water, images were acquired using an Dlympus FV-1000 confocal laser microscope (Dlympus Corporation, Tokyo, Japan). The Dil red D stain was then solubilized in isopropanol for 5 min and quantified spectrophotometrically at 490 nm using an XMark TM Microplate Spectrophotometer (Bio-Rad, CA, USA).

Nile Red staining
Cells were washed twice with ice-cold PBS and then fixed with 4% paraformaldehyde for 10 min at room temperature. After washing with PBS, the cells were stained with Nile Red (100 ng/mL) for 5 min in the dark at room temperature. Omages were acquired using an Dlympus FV-1000 confocal laser microscope and the fluorescence was quantified using Dlympus FV10-ASW 2.0 software.

Preparation of palmitate solution
Fatty acid-free bovine serum albumin (BSA)-conjugated palmitate solution was prepared. Palmitate stock solution (100 mM) dissolved in 100% ethanol by heating to 80°C was mixed with fatty acid-free BSA (2% in DMEM). The mixture was stirred at room temperature for 2 h and then diluted in culture media to give the final concentration indicated.

Measurement of reactive oxygen species
HepG2 cells seeded onto 35 mm cover glass-bottomed dishes (SPL Life Sciences, Seoul, Korea) were treated with 0.5 mM palmitate in the presence or absence of TENE for 24 h. The cells were incubated with 10 μM CM-H 2 DCF-DA for the final 30 min. Following two washes with ice-cold PBS, green fluorescence corresponding to the concentration of intracellular RDS was quantified using a 520 mm long-pass filter and an Dlympus FV-1000 laser fluorescence microscope.

Western blot analysis
Aliquots of whole cell lysates or tissue homogenates were subjected to SDS-polyacrylamide gel electrophoresis and the fractionated proteins were transferred onto Hybond-P + polyvinylidene difluoride membranes (GE Healthcare, Little Chalfont, UK). Membranes were blocked for 1 h at 37 °C with 5% non-fat milk in Tris-buffered saline (TBS) containing 0.1% Tween-20 and then incubated overnight at 4 °C with the indicated antibodies in TBS containing 0.1% Tween-20. Peroxidase-conjugated specific secondary antibody was then added to the membranes for 1 h at room temperature. After extensive washing in TBS containing 0.1% Tween-20, immunoreactive bands were detected using West-ZDL Plus (iNtRDN Biotechnology).

Animal study
All animal studies were performed in accordance with the Korean College of Laboratory Animal Medicine's Guidelines for Animal Care and Use, and were approved by the Onstitutional Animal Care and Use Committee of Konkuk University (approval numberI: KU15140). Male OCR mice were obtained from Drient Bio, Onc. (Seongnam, Korea) and housed at 21 ± 2 °C and 55 ± 5% humidity, with a 12 h light/dark cycle, and acclimatized to the facility for 7 days. Seven-week-old mice were then randomly assigned to one of four groupsI: a normal diet group (ND, containing 19.2% protein and 4.3% fat; Altromin Spezialfutter GmbH & Co., Lage, Germany), a HFD group (containing 24% protein and 24% fat; Research Diets, Onc., NJ, USA), a HFD plus TENE (300 mg/kg/day) group, and a ND plus TENE (300 mg/kg/day) group. TENE was administrated to the mice once daily by gavage for 8 weeks. After this period, the mice were euthanized and epididymal adipose tissue was excised and either frozen in liquid nitrogen or fixed in 4% paraformaldehyde solution for further analysis. Plasma total cholesterol, triglycerides, high-density lipoprotein (HDL), and low-density lipoprotein (LDL) were measured by Seoul Clinical Laboratories Services (Seoul, Korea).

Hematoxylin and Eosin staining
For histological examination of adipose tissue, epididymal adipose tissue fixed with 4% paraformaldehyde was embedded in paraffin. Paraffin blocks were cut into 4 μm slices, which were mounted on glass slides, deparaffinized, and rehydrated. The adipose sections were then stained with Hematoxylin and Eosin (H&E) and photographed under a light microscope.

Statistical analysis
Data are expressed as mean ± standard error (SE). Statistical significance was determined by ANDVA followed by post-hoc Bonferroni or Student's t-test, as appropriate. P < 0.05 was considered statistically significant.

Properties of TENE
To determine the physicochemical properties and bioavailability of nanoparticles for encapsulation, we analyzed the particle size and size distribution of TENE powder and TENE. The mean droplet size of TENE powder and TENE were 492.2 nm and 323.6 nm, respectively, indicating a single spectrum of size distribution. The curcumin content of TENE powder was 3.3 μg/mg, which was 15 times lower than that of turmeric extract powder (

Effects of TENE on intracellular lipid accumulation in 3T3-L1 adipocytes
To determine the optimal dose of TENE for 3T3-L1 cells, cell viability was assessed using the trypan blue exclusion method. When cells were incubated in the presence of a range of concentrations of TENE (0, 1, 2, 5, 10, or 30 μg/mL) for 24 h, a toxic effect of TENE was not observed up to 10 μg/mL; therefore, 10 μg/mL TENE was used in subsequent experiments ( Figure 1A). Because turmeric extract is a potential therapy for obesity (Ho et al., 2012;Septembre-Malaterre et al., 2016), we assessed whether TENE might affect adipogenesis in 3T3-L1 adipocytes. When 3T3-L1 cells were treated with MDO for 8 days, intracellular lipid content was significantly increased, according to Dil Red D staining. However, this increase in lipid content was significantly lower in the presence of TENE ( Figure 1B and 1C). Similar results were obtained when the cells were stained with Nile Red dye to quantify intracellular triglyceride ( Figure 1B and 1D). On line with these effects of TENE, polyphenols extracted from C. longa inhibit insulin-induced lipid accumulation and oxidative stress-related inflammatory response in 3T3-L1 adipocytes by regulating the expression of the adipokine adiponectin, and antioxidant enzymes, such as superoxide dismutase and catalase (Septembre-Malaterre et al., 2016). On addition, turmeric extracts exhibit powerful anti-obesity effects by stimulating lipolysis in 3T3-L1 adipocytes and animal models of obesity, in which they regulate glucose uptake, leptin secretion, and thus energy metabolism (Lee et al., 2010;Song & Choi, 2016). These findings are consistent with our data demonstrating the anti-adipogenic activity of TENE, which leads to reduced lipid accumulation in a cellular model. The data suggest that TENE may act by regulating cellular differentiation, which makes it suitable as a potential therapeutic agent for obesity (Ho et al., 2012;Song & Choi, 2016).

Effects of TENE on the expression of adipogenic genes in 3T3-L1 adipocytes
To dissect the mechanisms underlying the inhibitory effect of TENE on 3T3-L1 preadipocyte differentiation, the effect of TENE on the expression of the key adipogenic transcription factors PPAR-γ and C/EBP-α was evaluated. PPAR-γ and C/EBP-α protein levels dramatically increased following incubation with MDO solution for 8 days, whereas the levels of both proteins significantly lowered in the presence of TENE seeded in 60 mm culture dishes were grown to confluence and then stimulated with MDO for 2 days in the presence or absence of 10 μg/ml TENE. Cells were then incubated for an additional 4 days in complete medium containing 1 μg/ml insulin, and then followed incubation in complete medium for 2 days, replacing the medium every other day. After washing with PBS, the cells were fixed and stained with Dil Red D or Nile Red. Representative microscopic images are shown (B), and intracellular lipid content was assessed by measuring optical density at 490 nm (C) or fluorescence intensity (D). Bar, 100 μm. Data are expressed as mean ± S.E. (n=6). *p<0.01, **p<0.05 compared with the untreated group; #p<0.01 compared with the MDO-treated group.
( Figure 2A and 2B). Furthermore, TENE also suppressed the expression of aP2, a marker of mature adipocytes (Figure 2A and 2B), suggesting that TENE inhibits the adipogenic differentiation of 3T3-L1 preadipocytes by downregulating expression of the transcription factors that initiate adipogenesis. Control of PPAR-γ and C/EBP-α expression is the major regulatory mechanism involved in adipogenic differentiation (Cristancho & Lazar, 2011;Guo et al., 2015). Consistent with this concept, we have shown that MDO-induced adipogenic differentiation of 3T3-L1 cells is dramatically inhibited in the presence of TENE, which is associated with the downregulation of these key adipogenic factors. Although the biological effect of such turmeric extract-mediated anti-adipogenic activity has been documented in multiple reports (Lee et al., 2010;Septembre-Malaterre et al., 2016;Song & Choi, 2016), the effect of turmeric extract on the expression of PPAR-γ and C/EBP-α has not previously been shown. Additional studies on the anti-adipogenic effects of turmeric extract will be important to further elucidate the mechanisms underpinning its anti-obesity effects.

Effects of TENE on palmitate-induced lipid accumulation and ROS production in HepG2 cells
An imbalance in lipid synthesis and export in hepatocytes results in lipid accumulation and lipotoxicity (Leamy et al., 2014). Therefore, we examined the effects of TENE on the viability of HepG2 cells exposed to palmitate. While cell viability was dramatically reduced by palmitate treatment, this effect was ameliorated in a dose-dependent manner in HepG2 cells exposed to TENE ( Figure 3A).
To establish whether this TENE-mediated cytoprotection was due to suppression of lipid accumulation in HepG2 cells treated with palmitate, we measured the number and size of intracellular lipid vacuoles using Nile Red staining. Palmitate-induced lipid vacuoles were significantly fewer and smaller in the presence of 10 μg/mL TENE, indicating that TENE protects hepatocytes from palmitate-induced lipid accumulation ( Figure 3B and 3C).
The beneficial effects of turmeric extract on hepatic disease have also been demonstrated in the chemical-induced liver injury in which administration of turmeric extract was associated with a significant reduction in the hepatic oxidative states (Uchio et al., 2017;Lee et al., 2016). A dietary supplement containing turmeric extract also prevented the development of nonalcoholic fatty liver disease (NAFLD) by modulating the levels of serum glucose and leptin in patients with NAFLD (Navekar et al., 2017). These observations are in line with our present results demonstrating that TENE can improve palmitate-induced hepatotoxicity by regulating cellular accumulation of lipid.
To elucidate the mechanisms underlying the beneficial effect of TENE on cell viability, we also examined whether TENE affected the accumulation of intracellular RDS in HepG2 cells using the fluorescent probe H 2 DCF-DA. Palmitate significantly enhanced intracellular RDS production in HepG2 cells, whereas this was almost completely prevented by TENE (Figure 4). On line with this result, a hot water extract of turmeric inhibits ethanol-induced liver injury by inhibiting hepatic oxidative stress and production of inflammatory cytokines tumor necrosis factor (TNF)-α and interleukin (OL)-6 (Uchio et al., 2017). On addition, turmeric extract and its active compound curcumin also exhibited hepatoprotective activity against carbon tetrachloride-induced injury in mice by suppressing hepatic oxidative stress (Lee et al., 2016). These observations indicate that TENE may be effective in reducing lipid-or chemical-induced hepatocellular damage via anti-oxidative activity, in addition to its anti-adipogenic activity.

Effects of TENE on the expression of adipogenic genes and adipocyte size in mice fed a HFD
Because TENE downregulated the expression of the adipogenic transcription factors PPAR-γ and C/EBP-α during MDO-induced adipogenic differentiation of 3T3-L1 cells, we examined the effects of TENE in the epididymal adipose tissue of mice fed a HFD. Consistent with the above results, TENE significantly attenuated the HFD-induced expression of PPAR-γ and C/EBP-α.   adipogenic differentiation is associated with improved cellular energy metabolism in animal models of obesity (Lee et al., 2010;Septembre-Malaterre et al., 2016;Song & Choi, 2016). The effect of TENE on the regulation of adipogenic differentiation has not been fully investigated previously, but the present observations suggest that TENE contains anti-adipogenic compounds that may be beneficial for obese patients. TENE-mediated inhibition of adipogenic differentiation in adipose tissue may reduce the prevalence of associated metabolic disorders by improving the circulating lipid and cholesterol profile.

Conclusion
The present study shows that TENE significantly inhibited the adipogenic differentiation of 3T3-L1 preadipocytes by suppressing expression of PPAR-γ and C/EBP-α, thereby reducing intracellular lipid accumulation. Furthermore, oral administration of TENE to mice reduced the expression of these key adipogenic transcription factors in epididymal adipose tissue. These effects were accompanied by improvements in circulating lipid and cholesterol profile, indicative of whole body changes in lipid turnover. These results suggest that TENE has promising anti-adipogenic activity in both in vitro and in vivo models, which implies that it may have potential as an anti-obesity drug.
On addition, aP2 protein was also lower in epididymal adipose obtained from mice given TENE and a HFD versus mice fed a HFD alone ( Figure 5A and 5B). Furthermore, TENE also reduced the number and size of adipocytes in the epididymal adipose tissue ( Figure 5C), suggesting that it might reduce adiposity in HFD-fed obese mice by downregulating the expression of adipogenic and lipogenic genes induced by HFD.

Effects of TENE on the serum lipid profile of HFD-fed mice
To establish whether the anti-adipogenic effects of TENE were linked to changes in whole-body lipid metabolism, the effect of TENE on serum lipid profile was evaluated in the HFD-fed mice. The levels of triglyceride, total cholesterol, and low-density lipoprotein (LDL) were significantly increased in the serum of mice fed a HFD, compared with those of normal diet (ND)-fed mice, but these increases were significantly less in the presence of TENE (by 4.5%, 9.4%, and 9.9%, respectively) ( Figure 5D, 5E, and 5F). By contrast, oral administration of TENE to HFD-fed mice significantly ameliorated the HFD-induced suppression of high-density lipoprotein (HDL, by 6.2%) ( Figure 5G). Consistent with our observations, previous studies have reported that turmeric extract-mediated blockade of Figure 5. Effect of TENE on the expression of adipogenic genes, adipocyte size, and circulating lipid profile. Mice were fed with either a ND or HFD and administered with or without TENE for 8 weeks. (A and B) Lysates of epididymal adipose tissue were subjected to immunoblot analysis using indicated antibodies. Representative blots (A) and densitometric measurements are shown (B). (C) Epididymal adipose tissue was sectioned and the sizes of the adipocytes were analyzed by H&E staining. The sections were obtained from four independent experiments and representative images are shown. Bar, 50 μm. (D-G) Whole blood was obtained at the end-point of the experiment and serum was separated after sedimentation for 2 h at room temperature. The circulating concentrations of total cholesterol (D), triglyceride (E), LDL (F), and HDL (G) were analyzed and plotted. Data are mean ± S.E. (n=6). *p<0.01, **p<0.05 compared with the ND-fed group; #p<0.01, ##p<0.05 compared with the HFD-fed group.