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Introduction

Obesity is a significant public health concern that causes a high disease burden in developed countries (1). According to statistical data from 2015, obesity rates continue to rise worldwide, with 600 million adults and 100 million children reported to be obese in 195 countries (1). Moreover, obesity is a well-known risk factor for many chronic diseases such as type 2 diabetes, cardiovascular disease and degenerative brain disease (2). Obesity is caused by hereditary characteristics, endocrine disorders, dietary and environmental factors and an imbalance in energy intake and output (3). It is documented that abnormal fat accumulation in adipocytes, also called hypertrophic adipocytes, leads to the development of obesity. Hence, any material that suppresses excessive fat accumulation in adipocytes can be considered a potential anti-obesity agent.

Hypertrophic adipocytes are formed by the differentiation of pre-adipocytes into adipocytes or fat-laden cells, also known as adipogenesis. Preadipocyte differentiation is regulated by expression and activation (phosphorylation) of numerous transcription factors, enzymes and proteins. For example, the family members of peroxisome proliferator-activated receptors (PPARs) and CCAAT enhancer binding proteins (C/EBPs) are essential regulators of preadipocyte differentiation (4). Members of the signal transducer and activator of transcription (STAT) family are other important transcriptional factors that regulate preadipocyte differentiation (5,6). Notably, the expression and phosphorylation levels of these transcription factors increase significantly during adipogenesis (7). In addition, the expression of lipogenic enzymes such as fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC) and perilipin A, a lipid droplet (LD)-binding and stabilizing protein, is required for preadipocyte differentiation (8,9).

Lipolysis is the metabolic pathway through which lipids, mainly triglycerides (TG), are hydrolyzed into glycerol and free fatty acids and is controlled by a group of lipases, including hormone-sensitive lipase (HSL) expressed in adipocytes (10). Therefore, the promotion of lipolysis in lipid-laden adipocytes is regarded as another key strategy for preventing or treating obesity. A wealth of information strongly supports the link between chronic inflammation in adipose tissue (AT) and preadipocytes and the development of obesity (11). Tumor necrosis factor (TNF)-α is a pro-inflammatory cytokine that is expressed and secreted in preadipocytes (12). It has been revealed that TNF-α induces and aggravates inflammation in the AT (13). Cyclooxygenase-2 (COX-2) is an inducible and inflammatory enzyme responsible for the biosynthesis of prostaglandins (14). It is further documented that TNF-α is a strong inducer of COX-2 in various cells, including preadipocytes (15,16). Indeed, the authors of the present study as well as other groups have demonstrated that the short-term exposure of TNF-α leads to induction of COX-2 expression in 3T3-L1 preadipocytes (16-18). Thus, inhibition of the TNF-α-induced COX-2 expression in preadipocytes is considered as a possible target to manage obesity inflammation.

Nutmeg (Myristica fragrans) is a seed derived from an evergreen tree belonging to the Nutmegaceae family. It is a dioecious plant that grows to a height of 10-20 m and is cultivated in the Maluku province of Indonesia (19). Nutmeg is often used as a spice to enhance food flavor. Notably, nutmeg byproducts, such as seeds and mace, have been reported to have many potential bioactivities, including anti-obesity, anti-diabetic, anti-fungal, anti-microbial, antioxidant and anti-inflammatory (20-25). Evidence indicates that nutmeg seeds contain several alkylbenzene derivatives, such as myristicin, elemicin and safrole, which are toxic to the human organism (26,27). By contrast, lignans in nutmeg seeds are known to contain bioactive compounds, including nectandrin B, a nutmeg lignan with anti-aging and anti-diabetic effects (25,28). Interestingly, a previous study demonstrated that nutmeg lignans induce the IGF-1-AKT-mTOR pathway in an aging rat model, indicating their potential anti-sarcopenic effects (29). These findings illustrated that the nutmeg extraction method for concentrating bioactive lignan compounds, while removing toxic substances, has great potential for the treatment of various metabolic diseases. In previous studies, the authors prepared lignan-enriched nutmeg extract (LNX), which would comprise minimal levels of toxic myristicin (<0.5%) and maximum nectandrin B (28) and reported that LNX restores muscle proteins in aged mice (30), suggesting the potential of LNX as a therapeutic agent to overcome sarcopenia. However, the anti-obesity (antiadipogenic, prolipolytic and anti-inflammatory) effects of LNX on preadipocytes remain unclear. In the present study, the aim was to primarily demonstrate the LNX-contained whole active component's potential effects on inhibition of lipid accumulation to evaluate it as an anti-obesity drug candidate; therefore the authors focused on LNX, but not on nectandrin B. Therefore, the regulatory effects of LNX on lipid accumulation in differentiating preadipocytes, glycerol release and HSL phosphorylation in differentiated adipocytes and the TNF-α-induced COX-2 expression in preadipocytes by using 3T3-L1 cells, a mouse white preadipocyte cell line, were investigated. In the present study, it was reported that LNX exerts an anti-adipogenic effect on differentiating 3T3-L1 preadipocytes, which is mediated by the downregulating of STAT3 phosphorylation and FAS expression.

Materials and methods

Preparation of LNX

LNX used in the present study was obtained from Daehan Cell Pharm, Inc. (Guri, Republic of Korea). Briefly, the original seeds of 100 g nutmeg (Myristica fragrans) were pulverized and dissolved in 500 ml of 80% ethanol (EtOH). The optimal extraction conditions were determined by monitoring the minimal toxicity of myristicin and maximal bioactivity of nectandrin B. The extract was subjected to a mobile phase of the methanol (MeOH)-H2O (0-32 min, 63% MeOH; 32-37 min, 63-100% MeOH) mixture. The final extract was then adsorbed through Diaion HP-20 (Merck KGaA), one of the ion exchange resin, and eluted in a serial concentration of 30-90% EtOH to monitor the contents of myristicin and nectandrin B. The nutmeg alcohol extract was detected at 234 nm at a flow rate of 1.5 ml/min using a Waters HPLC Optima Pak C18 reverse-phase column (cat. no. OP C18-51002546; 4.6x250 mm, 5 µm pore size, RS Tech. Corp.). The mobile phase in the analytical column was composed of the mixture (the ratio of 4:6; absolute acetonitrile and 0.1% formic acid). In addition, nectandrin B concentration used in the present study is 4.2 from 91% of dry weight of LNX, which is lower than the concentration used in the previous study (28) with <0.5% of myristicin in a 91% of dry weight of LNX.

Chemicals and reagents

Primary antibodies against PPAR-γ (cat. no. sc-7272), C/EBP-α (cat. no. sc-61), phosphorylated (p)-STAT3 (cat. no. sc-8059) and total (T)-STAT3 (cat. no. sc-8019) were purchased from Santa Cruz Biotechnology, Inc. The primary antibody against FAS (cat. no. 9452) was obtained from BD Biosciences. Primary antibodies against p-HSL (S563) (cat. no. 4139) and p-HSL (S565) (cat. no. 4137) were acquired from Cell Signaling Technology, Inc. Primary antibodies against COX-2 (cat. no. 160106) and T-HSL (cat. no. 10006371) were purchased from Cayman Chemical Company. Primary antibody against perilipin A (cat. no. 3948-200) was purchased from BioVision, Inc. The primary antibody of β-actin (A5441), iso-butylmethylxanthine (IBMX), dexamethasone, insulin, Oil Red O solution, Isoproterenol, and Free Glycerol Reagent were obtained from MilliporeSigma. Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum (FBS), and penicillin/streptomycin were purchased from Welgene, Inc. Fetal calf serum (FCS) and trypan blue dye were purchased from Gibco; Thermo Fisher Scientific, Inc. Enhanced chemiluminescence (ECL) reagents were purchased from Advansta Inc.

Cell culture and differentiation

3T3-L1 mouse white preadipocytes (American Type Culture Collection) were cultured in DMEM containing 10% heat-inactivated FBS and 1% penicillin/streptomycin at 37˚C in a humidified atmosphere containing 5% CO2. 3T3-L1 preadipocytes were seeded with DMEM containing 10% FCS and 1% penicillin/streptomycin mixture and maintained up to the contact inhibition stage for 2 days. The differentiation of 3T3-L1 preadipocytes was initiated by replacing the media with new DMEM containing 10% heat-inactivated FBS plus a cocktail of hormones (MDI): 0.5 mM IBMX (M), 0.5 µM dexamethasone (D) and 5 µg/ml insulin (I) either with or without LNX at the designated concentrations for 2 days (Fig. 1A). After 2 days, the first differentiation medium was removed from the cells and the cells were subsequently cultured in new DMEM supplemented with 10% FBS and 5 µg/ml insulin either with or without LNX at the designated concentrations for an additional 3 days. After 3 days, the second differentiation medium was removed from the cells and the cells were further supplemented with new DMEM containing 10% FBS with or without LNX at the designated concentrations for another 3 days.

Figure 1 Effects of LNX on lipid accumulation during 3T3-L1 preadipocyte differentiation. (A) Experimental scheme for 3T3-L1 preadipocyte differentiation. (B) Measurement of intracellular LDs' accumulation in undifferentiated 3T3-L1 preadipocytes on D0 or differentiated adipocytes on D8 in the absence (control; 0.1% DMSO) or presence of LNX at the designated concentrations using Oil Red O staining. (C) Phase-contrast microscopic images are illustrated for the aforementioned conditions in panel B. (D) Cell survival assay was performed. (E) Quantification of Oil Red O staining images (B) to determine the percentage of LDs using ImageJ software. Data are presented as the mean ± SD (n=3). LNX, lignan-enriched nutmeg extract; LDs, lipid droplets; D, day; MDI; IBMX, dexamethasone and insulin.

Oil red O staining

On day 8 post the differentiation induction, the control or LNX-treated 3T3-L1 cells underwent phosphate-buffered saline (PBS) washing and 10% formaldehyde fixation for 2 h at room temperature (RT). Following a 60% isopropanol washing, the cells were thoroughly dried. The fixed cells were treated with Oil Red O working solution for 1 h at RT, followed by washing with distilled water. Light microscopy was used to identify the lipids or fats that accumulated in the conditioned cells (Nikon Corporation).

Cell count analysis

On day 8 post differentiation induction, control and LNX-treated 3T3-L1 cells were stained with 0.4% trypan blue dye at RT. Only cells with intact membranes could effectively block the dye. Once dead cells with damaged membranes were exposed to the dye, they were stained and counted under a light microscope.

Western blot analysis

At the designated time, the control or LNX-treated 3T3-L1 cells were washed with PBS and lysed in a modified RIPA buffer [50 mM Tris-Cl (pH 7.4), 150 mM NaCl, 0.1% SDS, 0.25% sodium deoxycholate, 1% Triton X-100, 1% Nonidet P-40, 1 mM EDTA, 1 mM EGTA, proteinase inhibitor cocktail (1X)]. The whole-cell lysates were collected and centrifuged at 14,000 x g for 15 min at 4°C. The supernatant was collected and protein concentrations were determined using bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific, Inc.).

An aliquot of protein (40 µg per lane) was separated using 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and then transferred to nitrocellulose membranes (MilliporeSigma). The membranes were washed with Tris-buffered saline (10 mM Tris, 150 mM NaCl) supplemented with 0.05% (v/v) Tween 20 (TBST) and blocked with blocking buffer (TBST containing 5% (w/v) non-fat dried milk) at 4˚C overnight. The membranes were incubated overnight with corresponding primary antibodies for C/EBP-α (1:2,000), PPAR-γ (1:2,000), p-STAT3 (1:2,000), STAT3 (1:2,000), phosphorylated (p)-STAT-5 (1:2,000), STAT-5 (1:2,000), FAS (1:1,000), perilipin A (1:2,000), p-HSL (S563) (1:2,000), p-HSL (S565) (1:2,000), T-HSL (1:2,000), COX-2 (1:2,000) or β-actin (1:10,000) at 4˚C. The membranes were then washed with TBST and incubated with a horseradish peroxidase-conjugated secondary antibody with either anti-mouse IgG (115-035-062; 1:5,000) or anti-rabbit IgG (111-035-045; 1:5,000) (Jackson ImmunoResearch Laboratories, Inc.) for 2 h at RT. The membranes were washed with TBST. ECL reagents were used to develop the images. Equal protein loading per lane was quantified based on the relative intensity of β-actin, an internal control protein. ImageJ software (v.1.6.0.24; National Institutes of Health) was used to intensify the bands for proteins that were standardized to the internal control.

Reverse transcription polymerase chain reaction (RT-PCR)

Total RNA was isolated from the control and LNX-treated 3T3-L1 cells after 4 h using RNAiso Plus (Takara Bio, Inc.). A total of three micrograms of total RNA were used to prepare complementary DNA using random hexadeoxynucleotide primers, reverse transcriptase (M-MLV RT), reverse transcriptase buffer (M-MLV RT 5X Buffer) and dNTPs (Promega Corporation). The cDNA was amplified by PCR with the following primers (Bioneer Corporation): COX-2 forward, 5'-TTGAAGACCAGGAGTACAGC-3' and reverse, 5'-GGTACAGTTCCATGACATCG-3'; and β-actin forward, 5'-TCATGAAGTGTGACGTTGACATCCGT-3' and reverse, 5'-CCTAGAAGCACTTGCGGTGCACGATG-3'. The PCR conditions applied for COX-2 were as follows; 30 cycles of denaturation at 95˚C for 30 sec, annealing at 62˚C for 30 sec and extension at 72˚C for 30 sec and for β-actin; 30 cycles of denaturation at 95˚C for 30 sec, annealing at 63˚C for 30 sec and extension at 72˚C for 1 min. Levels of β-actin mRNA expression were used as an internal control.

Quantification of glycerol content

On day 8 of the post-differentiation induction, 3T3-L1 cells were serum-starved for 2 h and further treated without or with LNX (6 µg/ml) or isoproterenol (ISO; 20 µM), a known lipolysis inducer for another 3 h. The culture medium from the control, LNX- or ISO-treated cells was stored and the glycerol content in the respective culture medium was measured using a Free Glycerol Reagent according to the manufacturer's instructions. The absorbance was measured at 540 nm using a microplate reader.

Statistical analysis

Cell counting, western blotting and RT-PCR analysis were conducted in triplicates and repeated two times. The mean and SD values were used to express the data. One-way ANOVA was employed to compare the significance of the differences, followed by a Bonferroni test for the post hoc analysis. P<0.05 was considered to indicate a statistically significant difference. The statistical software used in the present study was IBM SPSS Statistics 25 software (IBM Corp.).

Results

Treatment with LNX leads to a concentration-dependent suppression of lipid accumulation in differentiating 3T3-L1 preadipocytes

Initially, in the preliminary studies, LNX was tested at higher concentrations such as 50, 100 and 200 µg/ml for 3T3-L1 preadipocyte differentiation (data not shown). However, LNX treatment at 100 and 200 µg/ml had been demonstrating cytotoxicity in differentiating 3T3-L1 cells. Hence, LNX was tested in lower concentrations as revealed in the present study (6.25, 12.5 and 25 µg/ml) to further check the deposition rate of LDs during 3T3-L1 differentiation. Of interest, it was found that the accumulation of LDs appeared to be increased in the higher concentration of LNX in a dose-dependent manner (data not shown). Therefore, since the LNX effects on the inhibition of lipid accumulation during 3T3-L1 preadipocyte differentiation using Oil Red O staining had to be assessed, various concentrations of LNX (1.56, 3.12 and 6.25 µg/ml) have been selected for the present study. The timetable for 3T3-L1 pre-adipocyte differentiation used in the present study is depicted in Fig. 1A. As demonstrated in Fig. 1B, Oil Red O staining revealed markedly higher lipid accumulation in differentiated 3T3-L1 cells (D8) than in undifferentiated cells (D0). Notably, compared with mock-treated cells, treatment with LNX led to a dose-dependent inhibition of lipid accumulation in differentiated 3T3-L1 cells. The ability of LNX to downregulate lipid accumulation in differentiated 3T3-L1 cells was confirmed using phase-contrast microscopy (Fig. 1C). Additionally, the effects of 1.56, 3.12 or 6.25 µg/ml LNX on cell growth during the differentiation of 3T3-L1 preadipocytes into adipocytes, were examined by using cell count analysis. As illustrated in Fig. 1D, treatment with LNX at the tested doses did not significantly affect cell growth during 3T3-L1 preadipocyte differentiation. Quantification of the Oil Red O staining images to determine the LDs percentage using ImageJ software also revealed that LNX reduced lipid accumulation in a dose-dependent manner (Fig. 1E). Hence, based on the maximal lipid-lowering effect on differentiating 3T3-L1 preadipocytes with no cytotoxicity, the concentration of 6 µg/ml of LNX was selected for further experiments.

LNX treatment at 6 µg/ml selectively downregulates STAT3 phosphorylation and FAS expression in differentiating 3T3-L1 preadipocytes

Subsequently, to explore the fundamental molecular and cellular mechanisms underlying this LNX-induced anti-adipogenic effect, LNX (6 µg/ml) was investigated on the expression and phosphorylation levels of major adipogenic transcription factors, including C/EBP-α, PPAR-γ and STAT3/5, using immunoblotting analysis. As revealed in Fig. 2A, LNX treatment did not modulate significantly the expression levels of C/EBP-α and PPAR-γ proteins in 3T3-L1 preadipocyte differentiation at the assessed timepoints; however, it led to the slight elevation of the expression levels of C/EBP-α and PPAR-γ on D5 and D8, respectively. In addition, C/EBP-β was also assessed during 3T3-L1 differentiation. However, a band for this protein could not be obtained, which was possibly due to an antibody error.

Figure 2 Effects of LNX on the expression and/or phosphorylation levels of C/EBP-α, PPAR-γ, FAS, Perilipin A and STAT3 during 3T3-L1 preadipocyte differentiation. (A, C and D) 3T3-L1 preadipocytes were differentiated using induction medium containing IBMX, dexamethasone and insulin and FBS in the presence or absence of LNX and harvested at day 2, 5 and 8, respectively. Cellular proteins at the indicated time points were extracted and analyzed using western blot analysis. (B) Densitometric analysis of panel A. (C and D) Western blot analysis in triplicate experiments on D8. (E and F) The densitometric analysis of (C) and (D), respectively. *P<0.05 compared with vehicle control. LNX, lignan-enriched nutmeg extract; PPAR-γ, peroxisome proliferator-activated receptor gamma; C/EBP-α, CCAAT enhancer binding protein alpha; p-STAT3, phosphorylated signal transducer and activator of transcription 3; T-STAT3, total-STAT3; FAS, fatty acid synthase; D, day.

In the present study, the day courses such as D2, D5 and D8 in 3T3-L1 preadipocyte differentiation were observed to determine the p/T-STAT3 and FAS protein expression (Fig. 2A and B). It was revealed that STAT3 was highly phosphorylated during 3T3-L1 preadipocyte differentiation. Of note, p-STAT3 expression level was decreased on D5. In addition, on D8, STAT3 protein levels were strongly phosphorylated again during differentiation period (as compared with the control group of D5). Notably, the mid-phase of STAT3 decline affected the adipocyte differentiation. Meanwhile, FAS protein was greatly attenuated on D2 compared with D5 or D8. Hence, based on these day course experiment results, different day courses were investigated on each target (D8 for STAT3 and D2 for FAS protein expression levels) (Fig. 2C-F).

Interestingly, while treatment with LNX had no effect on the phosphorylation and total expression levels of STAT3 on D2 of 3T3-L1 cell differentiation, it markedly reduced the phosphorylation of STAT3 without altering its total expression levels on D5 of the cell differentiation. However, LNX treatment further reduced the phosphorylation and total expression levels of STAT3 on D8 of cell differentiation. Moreover, LNX treatment slightly inhibited the expression of FAS on D2, D5 and D8, during 3T3-L1 preadipocyte differentiation. LNX treatment did not influence perilipin A expression during cell differentiation. Expression levels of the control actin protein remained constant under these experimental conditions. The densitometric data from Fig. 2A are demonstrated in Fig. 2B. The results of the triplicate experiments, as demonstrated in Fig. 2C and D, revealed the capability of LNX (6 µg/ml) to vastly inhibit STAT3 phosphorylation and FAS expression on D8 and D2 of 3T3-L1 preadipocyte differentiation, respectively. The densitometric data from Fig. 2C and D are shown in Fig. 2E and F, respectively.

LNX treatment at 6 µg/ml does not stimulate lipolysis in differentiated 3T3-L1 adipocytes

Subsequently, it was explored whether treatment with LNX (6 µg/ml) modulated lipolysis in differentiated 3T3-L1 adipocytes by measuring the levels of glycerol content and HSL phosphorylation, two hallmarks of lipolysis. The experimental scheme and timescale for measuring glycerol content and HSL phosphorylation are exhibited in Fig. 3A. To this end, differentiated 3T3-L1 adipocytes on D8 were serum-starved (0% FBS) for 2 h and incubated in a fresh culture media containing 10% FBS in the absence or presence of LNX (6 µg/ml) or ISO (20 µM), a known inducer of lipolysis (31), for an additional 3 h. Culture media and whole-cell lysates from each condition were used to measure the two lipolysis hallmarks aforementioned. As illustrated in Fig. 3B, ISO treatment for 3 h significantly enhanced glycerol release from differentiated 3T3-L1 adipocytes, whereas LNX treatment did not. In addition, while ISO treatment for 3 h significantly elevated p-HSL levels at residues S563 and S565 in differentiated 3T3-L1 adipocytes, LNX treatment had no stimulatory effect. The total HSL protein expression levels remained unchanged under these experimental conditions. The densitometric data from Fig. 3C are demonstrated in Fig. 3D.

Figure 3 Effects of LNX on glycerol release and the phosphorylation levels of HSL in differentiated adipocytes. (A) The experimental scheme for measurement of glycerol content and HSL phosphorylation in differentiated 3T3-L1 cells. (B) Differentiated 3T3-L1 cells on D8 (0 h) were serum-starved for 2 h and then cultured in the absence (control; 0.1% DMSO) or presence of LNX (6 µg/ml) or ISO (20 µM) for an additional 3 h. Glycerol content in culture medium from control or drug (LNX or ISO)-treated cells was analyzed in triplicates. Data are presented as the mean ± SD of three independent experiments. *P<0.05 vs. control. (C) After the treatment aforementioned, whole-cell lysates were extracted and analyzed using western blot analysis with corresponding antibodies. (D) The densitometric analysis of panel C. LNX, lignan-enriched nutmeg extract; HSL, hormone-sensitive lipase; ISO, isoproterenol; p, phosphorylated; T, total.

LNX treatment has no effect on the TNF-α-induced expression of COX-2 in 3T3-L1 preadipocytes

Next, it was further explored whether TNF-α at 10 ng/ml induces COX-2 expression in 3T3-L1 preadipocytes and whether treatment of LNX at different concentrations (1.5, 3 or 6 µg/ml) interferes with it by using RT-PCR analysis. As expected, the exposure of TNF-α for 4 h greatly induced COX-2 mRNA expression in 3T3-L1 preadipocytes. However, LNX treatment at the examined concentrations did not significantly influence cytokine-induced COX-2 mRNA expression in these cells (Fig. S1). Using immunoblotting analysis (Fig. 4A), it was investigated whether LNX treatment at higher doses (10, 25 and 50 µg/ml) could modulate cytokine-induced COX-2 expression in 3T3-L1 preadipocytes. As revealed in Fig. 4, while treatment with TNF-α for 4 h markedly enhanced COX-2 protein expression in 3T3-L1 preadipocytes, LNX treatment at such high doses tested did not interfere with the TNF-α-induced COX-2 protein expression in 3T3-L1 preadipocytes. Expression levels of the control actin protein remained constant under these experimental conditions. The densitometric data from Fig. 4A are illustrated in Fig. 4B.

Figure 4 Effects of LNX treatment on TNF-α-induced COX-2 protein expression level in 3T3-L1 preadipocytes. (A) 3T3-L1 preadipocytes were treated with or without TNF-α (10 ng/ml) in the presence or absence of LNX at the designated concentrations for 4 h. After the aforementioned treatment, whole-cell lysate was prepared and analyzed using western blotting. (B) The densitometric analysis of panel A. LNX, lignan-enriched nutmeg extract; COX-2, cyclooxygenase-2.

Discussion

Abnormal lipid accumulation, aggravated inflammation and the dysregulation of preadipocyte lipolysis are closely linked to the development of obesity. In the present study, LNX demonstrated anti-adipogenic, but not pro-lipolytic, and anti-inflammatory effects in differentiating 3T3-L1 preadipocytes through the control of STAT3 phosphorylation and FAS expression.

In the initial experiments, it was revealed that LNX treatment led to a concentration-dependent suppression of fat accumulation in differentiating 3T3-L1 preadipocytes with no cytotoxicity, supporting its anti-adipogenic effect. Mounting evidence illustrated a pivotal role of adipogenic transcription factors including C/EBP-α, PPAR-γ and STAT3/5 in adipogenesis of preadipocytes (32-34). To date, LNX regulation of the expression and phosphorylation levels of these transcription factors during the adipocyte differentiation of 3T3-L1 preadipocytes remains unknown. In the present study, LNX treatment greatly inhibited STAT3 phosphorylation without affecting total protein expression in the middle stage (D5) of cell differentiation. Previous studies have shown that STAT3 phosphorylation is involved in the early stages of 3T3-L1 preadipocyte differentiation (35,36). These findings suggested that LNX exerts its lipid-lowering effect on differentiating 3T3-L1 preadipocytes by inhibiting STAT3 and its downstream components or pathways. Previously, it has also been reported that STAT3 regulates the upregulation of C/EBP-α and PPAR-γ at middle or last stage of preadipocyte differentiation (37). However, in the present study, it was demonstrated that LNX treatment does not modulate the expression levels of both C/EBP-α and PPAR-γ throughout 3T3-L1 preadipocyte differentiation. It is therefore conceivable that the anti-adipogenic effect of LNX on differentiating 3T3-L1 preadipocytes is mediated not through the STAT3-dependent C/EBP-α and PPAR-γ pathway but via a different STAT3-dependent pathway.

Preadipocyte differentiation is also closely associated with lipogenesis and lipid or LD stabilization (38,39). FAS is a key lipogenic enzyme responsible for catalyzing de novo synthesis of fatty acids and its expression is highly elevated during preadipocyte differentiation (40,41). Perilipin A is an LD-binding and stabilizing protein whose expression is greatly enhanced during preadipocyte differentiation (42,43). At present, little is known about the LNX regulation of FAS and perilipin A during preadipocyte differentiation. Notably, LNX treatment inhibited FAS expression in the early stages of 3T3-L1 preadipocyte differentiation. However, the present study demonstrated that LNX treatment does not interfere with perilipin A expression during 3T3-L1 cell differentiation. These results suggested that the anti-adipogenic effect of LNX on differentiating 3T3-L1 preadipocytes is, in part, mediated through interference with FAS-dependent lipogenesis.

Lipolysis is a biochemical process in which lipids or TGs stored in adipocytes are broken down, resulting in the efflux of fatty acids and glycerol (44). Hence, any material that induces lipolysis in lipid-laden adipocytes can be utilized as a potential anti-obesity agent. Accordingly, the LNX regulation of lipolysis and its regulator in differentiated or mature adipocytes were further tested. However, considering the present findings that LNX treatment did not enhance glycerol release in differentiated 3T3-L1 adipocytes, it is likely that LNX has no lipolytic activity in mature 3T3-L1 adipocytes. Cellular lipases which trigger lipolysis HSL are key for TG degradation in mature adipocytes (45). It has also been revealed that HSL is phosphorylated at S563 and S565 and becomes active in adipocytes in response to certain endogenous or exogenous stimuli, such as norepinephrine or ISO (46,47). Given that, unlike ISO, LNX treatment does not induce the phosphorylation of HSL at residues S563 and S565 in differentiated 3T3-L1 adipocytes, it is further evident that LNX has no lipolytic effect on these cells.

Accumulating evidence strongly indicates that chronic inflammation contributes to development of obesity (48). A wealth of information illustrates that preadipocytes, the predominant cell types present in the AT, are able to express and secrete numerous pro-inflammatory cytokines [including TNF-α, interleukin (IL)-1β and IL-6] and enzymes (COX-2) (49-51), which can confer and aggravate obese inflammation, via the autocrine, paracrine and endocrine ways. As aforementioned, the authors and other groups have previously demonstrated that TNF-α is an inducer of COX-2 in not only 3T3-L1 preadipocytes but also in mature or differentiated adipocytes (15-19). To date, the regulation of pro-inflammatory cytokine-induced expression of COX-2 by LNX in 3T3-L1 preadipocytes remains unknown. In the present study, it was observed that LNX treatment at doses tested does not interfere with the TNF-α-induced COX-2 expression at the protein and mRNA levels in undifferentiated 3T3-L1 cells. These results indicated that LNX has no anti-inflammatory effect on 3T3-L1 preadipocytes. In addition, it would be interesting to investigate whether LNX could alter the cytokine-induced expression of COX-2 (and other inflammatory mediators) in mature adipocytes. Moreover, further studies using in vivo experiments will be able to address the limitations of this study.

In summary, it was firstly reported that LNX has a potent anti-adipogenic effect on differentiating 3T3-L1 preadipocytes, and this effect is achieved by downregulating STAT3 phosphorylation and FAS expression. The present study advocated that LNX may be a potential obesity-preventive agent.

Supplementary Material

Effects of LNX on TNF-α-induced COX-2 mRNA expression level in 3T3-L1 preadipocytes. (A) 3T3-L1 preadipocytes were treated with or without TNF-α (10 ng/ml) in the presence or absence of LNX at the designated concentrations for 4 h. After the aforementioned treatment, total RNA was extracted and analyzed for COX-2 using reverse transcription PCR analysis. (B) The densitometric analysis of panel A. LNX, lignan-enriched nutmeg extract; COX-2, cyclooxygenase-2.

Acknowledgements

The authors would like to thank Daehan-Nupharm Co., Ltd. (Hwasung, Republic of Korea) for providing the nutmeg extract samples and Geron Biotech Ltd., KBSI Research Company, for the help and support on the current publication.

Funding

Funding: The present study was supported by the Korea Basic Science Institute (grant. no. C280320).

Availability of data and materials

All data generated or analyzed during this study are included in this published article.

Authors' contributions

NP, SD, JL, HY, BJ and JC conceived and designed the experiments. NP, SD, GB, JL, GK and AM performed the experiments. HY, BJ, JC, SD, GB and AM analyzed and interpreted the data. HY, BJ, AM and JC wrote the manuscript; BJ, JL, GB and AM reviewed and edited the manuscript. All authors read and approved the final version of the manuscript. HY, BJ and JC confirm the authenticity of all the raw data.

Ethics approval and consent to participate

Not applicable.

Patient consent for publication

Not applicable.

Competing interests

The authors declare that they have no competing interests.

References