Piperine, a component of black pepper, decreases eugenol-induced cAMP and calcium levels in non-chemosensory 3T3-L1 cells

Highlights • An ethanol extract of black pepper inhibits eugenol-induced signal transduction.• The extract reduces eugenol-induced cAMP and Ca2+ levels, and phosphorylation of CREB.• Piperine inhibits eugenol-induced signal transduction.• Piperine reduces eugenol-induced cAMP and Ca2+ levels, and phosphorylation of CREB.


Introduction
Black pepper (Piper nigrum, PN), of the Piperaceae family, is one of the most widely used condiments in the world. In addition to its use as a spice, black pepper has been used in conventional medicines to treat pain, flu, muscle aches, and rheumatism as well as to stimulate appetite [1]. Black pepper contains various active phytochemicals such as alkaloids, terpenes, flavones, and steroids, and many studies have reported their physiological effects in human and animals [1,2]. Although the exact amount of piperine, a main alkaloid in black pepper, varies owing to different methods of extraction and analysis, piperine comprises approximately 2-30% of dried black pepper [3,4]. After piperine (Fig. 2B) was first separated and purified by Oersted in 1820 [5], numerous studies have demonstrated its potential health benefits [1,2]. Piperine is an anti-inflammatory molecule inhibiting the production of prostaglandin E2 and nitric oxide in RAW264.7 cells [6]. It also suppresses stress-induced behavior by increasing serotonin and brain-derived neurotropic factors [7]. In high-fat diet-induced obese mice, piperine was shown to activate AMP-activated protein kinase and PPARd and attenuate HFD-induced obesity [8]. Inhibition of ERK1/2 signaling by piperine reduced SREBP-1 and FAS expression in breast cancer cells, suggesting that it could be used as an antitumor agent to prevent or treat human breast cancer [9]. Recently, this possibility was further supported by data showing that piperine induced apoptosis of melanoma cells by decreasing XIAP, Bid, and caspase-3 [10]. However, the physiological roles of black pepper and piperine have not yet been elucidated in the odorant-induced signal transduction (OST) pathway in nonchemosensory cells.
Olfaction is the sense of smell. The perception of odor is important for survival and is required to select food and mates as well as to respond to the fear of predators. However, recent reports demonstrate that OST plays not only a role in olfaction but also other physiological roles in non-chemosensory tissues [11]. Ectopic expressions of olfactory receptors in sperm, kidney, and muscle were involved in the chemotaxis of sperm, glomerular filtration rate, and muscle regeneration, respectively [12][13][14]. The ectopic expression was also supported by recent reports showing that olfactory receptors were expressed in fat tissue of diet-induced obese mice and eugenol receptor (mOR-EG, Olfr73, MOR174-9) was expressed in 3T3-L1 cells [15,16]. However, their physiological roles and regulations in fat tissue and non-chemosensory cells are Abbreviations: CREB, cAMP response element-binding protein; OST, odorant-induced signal transduction; PNF, Piper nigrum fructus largely unknown. Interestingly, the OST pathway in these nonchemosensory tissues shares the same mechanism, where odorants stimulate signals by binding to olfactory receptors, and cAMP and Ca 2+ act as second messengers to relay the signal cascade in order to achieve olfactory perception and other physiological effects in neuronal and non-chemosensory cells, respectively [12][13][14].
In this study, we investigated the effects of black pepper and its constituent, piperine, on the OST pathway in non-chemosensory 3T3-L1 cells. When an odorant was used to stimulate non-chemosensory cells, we observed inhibitory effects of the ethanol extract of black pepper and piperine through regulation of Ca 2+ and cAMP levels.

Plant material
Ethanol extract of P. nigrum fructus (PNF) was obtained from the Korea plant extract bank at the Korea Research Institute of Bioscience and Biotechnology (KRIBB, Daejeon, Republic of Korea). The powder was dried at room temperature and then dissolved in 95% ethanol (v/v).

Reagents and antibodies
Piperine and eugenol were purchased from Sigma (St. Louis, MO, USA). The Ca 2+ assay kit was obtained from Molecular Devices (Sunnyvale, CA, USA). The cAMP assay kit was purchased from Enzo Life Sciences (Plymouth Meeting, PA, USA). Antibodies against phospho-CREB and lamin B1 were purchased from Cell Signaling Technology (Beverly, MA, USA).

Cell culture and viability assay
3T3-L1 cells were obtained from American Type Culture Collection (Manassas, VA, USA) and cultured in DMEM (high glucose), which contained 10% FBS and 1Â antibiotic-antimycotic solution (WelGENE Inc., Daegu, Republic of Korea). The cells were incubated at 37°C in the presence of 5% CO 2 . The viability of cells was determined using the Cell Proliferation Reagent WST-1 (Roche Diagnostics, Mannheim, Germany). Cells at a concentration of 4 Â 10 4 cells/well in 500 ll culture medium were seeded in 24well plate. Incubated cells for 6 h at 37°C and 5% CO2 and washed it with serum-free medium, then added 500 ll serum-free medium containing the extract or piperine. After 24 h, added 20 ll/well Cell Proliferation Reagent WST-1 and incubate the cells for 1 h. Shake thoroughly for 1 min on a shaker and measure the absorbance of the samples against a background control as blank using the microplate reader at 440 nm and 690 nm.

UPLC-MS/MS analysis
The analyses were performed using an Acquity UPLC system (Waters, Milford, MA, USA) with an Acquity UPLC BEH C18 column (2.1 mm Â 100 mm, 1.7 lM). The eluent was a gradient of 0.1% formic acid aqueous solution (Solvent A) and 0.1% formic acid in methanol (Solvent B) at a flow rate of 0.4 mL/min. The cells were initially treated with 98% A, followed by 98-25% A for 0-5 min, 25% A for 5-6 min, and finally 98% A for 6-7 min, which was held  for 1 min. The injection volume was 2 ll, the column temperature was kept at 35°C and the total run time was 8 min. The identification and quantification of piperine for P. nigrum fructus was carried out using a Waters Xevo TQ triple-quadrupole mass spectrometer equipped with an electrospray ionization mode. The tandem MS was operated in positive ESI mode, and the data was processed using MassLynx 4.1 (Waters) software and the multiple re-action monitoring mode. The detector was operated at a cone voltage of 32 V and a capillary voltage of 2.7 kV. The source temperature was set at 150°C, while the desolvation flow was set at 800 L/h; the desolvation gas temperature was set at 400°C.

Ca 2+ assay
3T3-L1 cells were seeded in 96-well black plates and pretreated with PNF and piperine in a CO 2 incubator for 30 min before adding 100 ll of component A buffer (Molecular Devices, Sunnyvale, CA, USA). The plate was covered with foil and incubated for 30 min at room temperature, followed by 15 min at 37°C. Eugenol and ionomycin were added in Flexstation 3 (Molecular Devices). The Ca 2+ level was measured according to the manufacturer's instructions.

cAMP assay
Before treatment with PNF and piperine, the 3T3 L1 cells were starved with serum-free DMEM (high glucose) media for 16-18 h. After 30 min of pretreatment with PNF and piperine, the cells were treated with eugenol for 7 min and then lysed with 0.1 M HCl. cAMP levels were measured using the Direct cAMP EIA Kit (Enzo Life Sciences).

Western blot analysis
Cells were seeded in 6-well plates and after 1 day, they were starved with serum-free DMEM for 16   A total of 40 lg of the protein was electrophoresed by SDS-PAGE and transferred to a nitrocellulose membrane. The membranes were incubated overnight with the suitable primary antibody at 4°C. The membranes were probed with HPR-conjugated secondary antibody at room temperature for 1 h, and then they were incubated with enhanced chemiluminescence reagents (Amersham, Piscataway, NJ, USA).

Statistical analysis
All experiments were repeated at least three times, and the data are expressed as the mean ± standard deviation (SD). Group means were compared using the non-parametric Kruskal-Wallis and Mann-Whitney analysis using SPSS (SPSS Inc., Armonk, NY).

PNF decreased Ca 2+ , cAMP levels in 3T3-L1 cells
In our previous findings, Ca 2+ and cAMP levels were shown to increase with eugenol treatment in 3T3-L1 cells [16]. Our current investigation explores the effects of PNF on the eugenol-induced signal transduction pathway in 3T3-L1 cells. As shown in Fig. 1A, lower concentrations of PNF (25-50 lg/mL) had little effect on Ca 2+ levels. However, Ca 2+ levels were significantly decreased up to 40% with 100 lg/mL of PNF and more than 70% with 200 lg/mL of PNF. Since cAMP is an important molecule that affects Ca 2+ levels in odorant signaling transduction (OST), we determined cAMP levels after treatment with PNF. cAMP levels were gradually reduced from an initial concentration of 25 lg/mL to nearly 50% of that after treatment with 100 lg/mL of PNF (Fig. 1B). We observed a difference in eugenol-responsiveness as determined with cAMP and Ca 2+ assay, which may due to cell type and receptor-coupled G proteins as previously described by Touhara [17]. Responsiveness of Ca 2+ and cAMP have different patterns with the stimulation of the same odorants in HKE293 cells [18] and HeLa/Olf cells [19]. These suggest that change of Ca 2+ does not fully reflect change of cAMP. Instead, changes of Ca 2+ and cAMP mutually represent activation or inhibition of OST. Our data suggest that PNF inhibits the OST pathway through regulation of Ca 2+ and cAMP levels induced by eugenol in 3T3-L1 cells. To remove the possibility of toxic effect of the extract on the change of Ca 2+ and cAMP, the cell viability was measured by WST-1 assay.
As shown in Fig. 1C, up to 800 lg/mL treatment with PNF for 30 min did not inhibit 3T3-L1 proliferation. It suggests that the change of cAMP and Ca 2+ is not caused by toxicity.

The phosphorylation of CREB was decreased by PNF in 3T3-L1 cells
To test whether PNF also decreases signal molecules as a result of decreasing Ca 2+ and cAMP levels in 3T3-L1 cells, we investigated the phosphorylation of CREB, known to be regulated by cAMP levels in the OST signaling pathway [20]. Phosphorylation of CREB by eugenol was decreased in a dose-dependent manner and remarkably decreased after 30 min of pre-treatment with 200 lg/mL of PNF (Fig. 1D). This result suggests that cAMP levels decreased by PNF affect the phosphorylation of CREB.

Determination of piperine in the ethanol extract of black pepper using UPLC-MS/MS
Since piperine is a major active alkaloid in black pepper [21], UPLC-MS/MS was used to evaluate its concentration in an extract of black pepper. Under the chromatographic conditions described above, the retention time of piperine was 5.5 min ( Fig. 2A) and its molecular mass was 285 Da. To further characterize the mass peak, the peak was analyzed using a mass spectrometer. The parent fragmentation ions were m/z 286.5 [M+H] + for piperine. The parent-to-daughter ion transitions were monitored at m/z 286.5 ? 201.3, 135.2 (Fig. 2B). The piperine content was 133.8 ± 17.6 mg/g (in the ethanol extract).

Reduction of odorant-induced Ca 2+ and cAMP levels by piperine in 3T3-L1 cells
Because PNF inhibited the OST pathway and piperine is one of its components in the ethanol extract, we examined the effects of piperine on the OST pathway in 3T3-L1 cells. First, it was found that increased Ca 2+ levels in response to eugenol began to decrease with pretreatment of 400 lM piperine for 30 min and was further decreased by 75% with 800 lM (Fig. 3A). In addition, cAMP levels also decreased up to 50% with 30 min of pretreatment with piperine (100, 200, 400, and 800 lM) (Fig. 3B). These results showed that piperine inhibits the OST pathway by regulating Ca 2+ and cAMP levels in 3T3-L1 cells as PNF did. Again, we observed the difference in eugenol-responsiveness by piperine in cAMP and Ca 2+ assay as previously described in Fig. 1. However, decreases of both cAMP and Ca 2+ level by piperine represent the inhibition of eugenol-induced signal transduction in 3T3-L1 cells. In addition, it strongly suggests that in the ethanol extract of black pepper, piperine is at least one of the active components to regulate eugenol-induced signal transduction. To whether the change of cAMP and Ca 2+ was due to toxicity of piperine, the cell viability was determined. As shown in Fig. 3C, piperine (100-800 lM) did not inhibit cell proliferation. It suggests that the change of cAMP and Ca 2+ is not caused by toxic effect of piperine on 3T3-L1 cells.

Piperine suppressed phosphorylation of CREB in 3T3-L1 cells
Next, we investigated whether the upregulated phosphorylation of CREB by eugenol was decreased by piperine as it was with PNF. As shown in Fig. 3D, piperine significantly decreased the eugenol-induced CREB phosphorylation in 3T3-L1 cells. This is consistent with the previously observed effects of piperine and PNF on the OST pathway. It further strengthens the previous conclusion that piperine is one of the active molecules inhibiting eugenolinduced signal transduction in the ethanol extract of black pepper.
In summary, our study showed that pretreatment with PNF and piperine modulated Ca 2+ influx, cAMP levels, and phosphorylation of CREB in response to eugenol in non-chemosensory 3T3-L1 cells. These findings also suggest that mOR-EG is at least one of mediators responding to eugenol in 3T3-L1 cells. However, further study will be required to investigate the physiological roles of eugenol receptor in 3T3-L1 cells.