PipeNig®-FL, a Fluid Extract of Black Pepper (Piper Nigrum L.) with a High Standardized Content of Trans-β-Caryophyllene, Reduces Lipid Accumulation in 3T3-L1 Preadipocytes and Improves Glucose Uptake in C2C12 Myotubes

Abstract

Trans-β-caryophyllene (BCP) is a natural sesquiterpene hydrocarbon with several important pharmacological activities, including antioxidant, anti-inflammatory, anticancer, and cardioprotective functions. These properties are mainly due to its selective interaction with the peripherally expressed cannabinoid receptor 2. In addition, BCP activates peroxisome proliferated activator receptors α and γ and inhibits the Toll-like receptor signaling pathway. Given the growing scientific interest in BCP, the aim of our study was to investigate the metabolic effects of a black pepper extract (PipeNig^®-FL), containing a high standardized content of BCP. In particular our interest was focused on its potential activity on lipid accumulation and glucose uptake. The extract PipeNig^®-FL was chemically characterized by gas chromatography-mass spectrometry (GC-MS) and gas chromatography with flame-ionization detection (GC-FID), confirming a high content (814 mg/g) of BCP. Experiments were performed on 3T3-L1 preadipocytes and on C2C12 myotubes. Lipid content following 3T3-L1 adipogenic differentiation was quantified with AdipoRed fluorescence staining. Glucose uptake and GLUT4 membrane translocation were studied in C2C12 myotubes with the fluorescent glucose analog 2-NBDG and by immunofluorescence analysis. Here we show that PipeNig^®-FL reduces 3T3-L1 adipocyte differentiation and lipid accumulation. Moreover, acute exposure of C2C12 myotubes to PipeNig^®-FL improves glucose uptake activity and GLUT4 migration. Taken together, these results reveal interesting and novel properties of BCP, suggesting potential applications in the prevention of lipid accumulation and in the improvement of glucose uptake.

Keywords: GLUT4; adipogenesis; black pepper; glucose uptake; lipid accumulation; trans-β-caryophyllene.

Figure 1

Gas chromatography–mass spectrometry (GC–MS) total ion current gas-chromatogram of PipeNig^®-FL. The main compound trans-β-caryophyllene (BCP) is out of scale in order to evidence the other minor monoterpenes and sesquiterpenes that characterize the chemical composition of PipeNig^®-FL. The inset shows the chemical formula and the mass spectrum of BCP. The y axis is the total ion current; the x axis represents time (in min).

Figure 2

PipeNig^®-FL affects 3T3-L1 cell viability only at high (millimolar) concentrations. 3T3-L1 cells were induced to differentiate into adipocytes for 9 days and treated with increasing concentrations of PipeNig^®-FL for the entire differentiation period. The bar graph summarizes cell viability based on ATP content. Data in percentage referred to control condition are represented as the mean ± standard error of the mean (SEM) of three independent experiments. *** p < 0.001 vs. control.

Figure 3

PipeNig^®-FL reduces intracellular lipid accumulation in 3T3-L1 cells without altering the cell number. (A) Representative images of AdipoRed (red) and NucBlue (blue) staining of undifferentiated preadipocytes (UNDIFF), differentiated control adipocytes (CTRL) and 10 µM PipeNig^®-FL-treated 3T3-L1 adipocytes after 9 days of differentiation. Scale bar 50 µm. (B) Bar graph summarizing AdipoRed staining experiments to assess lipid accumulation on undifferentiated cells, differentiated control and 3T3-L1 adipocytes treated with various concentrations of PipeNig^®-FL for 9 days, showing triglyceride accumulation per well. (C) Bar graph summarizing NucBlue staining experiments to assess variations in the number of cells, showing DNA content per well. (D) Bar graph showing triglyceride accumulation per cell, calculated as the ratio of AdipoRed and NucBlue staining. Data in percentage referred to differentiated control condition are represented as the mean ± SEM of three independent experiments. * p < 0.05; ** p < 0.01 vs. control.

Figure 4

PipeNig^®-FL does not have any effects on cell viability in C2C12 muscle cell. C2C12 cells were treated with increasing concentration of PipeNig^®-FL for 1h. Data in percentage referred to control condition are represented as the mean ± SEM (n = 3).

Figure 5

PipeNig^®-FL stimulates glucose uptake. (A) Representative confocal images of C2C12 myotubes incubated with the fluorescent glucose analog 2-NBDG for 30 min. Images are presented in pseudocolor (LUT = fire) to better show the fluorescence intensity variations. Insulin (25 nm) was used as a positive control. Scale bar 36 µm. (B) Bar graph summarizing the experiments of fluorescent glucose uptake. Data in percentage referred to control condition are represented as the mean ± SEM (n = 4). * p < 0.05; ** p < 0.01 vs. control.

Figure 6

PipeNig^®-FL induces GLUT4 translocation to the plasma membrane. (A) Confocal images of a representative experiment of GLUT4 immunofluorescence staining. After PipeNig^®-FL stimulation (1–10–100 nM) the fluorescent signal is clearly localized to the peripheral plasmalemma, thus suggesting the GLUT4 translocation. Images are presented in pseudocolor (LUT = fire) to better show the fluorescence intensity variations. Insulin (25 nM) was used as a positive control. Scale bar 36 µm. (B) Bar graph representing the ratio peripheral vs. internal GLUT4 fluorescence intensity. Data are represented as the mean ± SEM of three independent experiments. * p < 0.05; ** p < 0.01; *** p < 0,001 vs. control.