A Citrus bergamia Extract Decreases Adipogenesis and Increases Lipolysis by Modulating PPAR Levels in Mesenchymal Stem Cells from Human Adipose Tissue

Abstract

The aim of this research was to assess the impact of a well-characterized extract from Citrus bergamia juice on adipogenesis and/or lipolysis using mesenchymal stem cells from human adipose tissue as a cell model. To evaluate the effects on adipogenesis, some cell cultures were treated with adipogenic medium plus 10 or 100 μg/mL of extract. To determine the properties on lipolysis, additional mesenchymal stem cells were cultured with adipogenic medium for 14 days and after this time added with Citrus bergamia for further 14 days. To verify adipogenic differentiation, oil red O staining at 7, 14, 21, and 28 days was performed. Moreover, the expression of peroxisome proliferator-activated receptor gamma (PPAR-γ), adipocytes fatty acid-binding protein (A-FABP), adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), monoglyceride lipase (MGL), 5'-adenosine monophosphate-activated protein kinase (AMPK)α1/2, and pAMPKα1/2 was evaluated by Western blot analysis and the release of glycerol by colorimetric assay. Citrus bergamia extract suppressed the accumulation of intracellular lipids in mesenchymal stem cells during adipogenic differentiation and promoted lipolysis by repressing the expression of adipogenic genes and activating lipolytic genes. Citrus bergamia extract could be a useful natural product for improving adipose mobilization in obesity-related disorders.

Figure 1

Effects of Citrus bergamia extract (CBE) on mesenchymal stem cells viability, determined using the 3-(4,5-dimethyl-2 thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay, after 48 hours of treatment with 1, 10, 50, or 100 μg/mL of CBE. The values of optical density measured at λ = 550 nm are reported as percentage with respect to the optical density registered for untreated control (Control), the latter considered as 100% of cell viability. The values are mean ± SD of three experiments performed in triplicate.

Figure 2

Effect of 10 and 100 μg/mL Citrus bergamia extract (CBE) on the lipid content of mesenchymal stem cells stained with oil red O during adipogenic differentiation. Cells were examined at 7, 14, 21, and 28 days from the start of differentiation. (a) A representative photograph from three independent experiments of untreated control (Control), adipogenic medium-treated cells (AM), and AM plus 10 or 100 μg/mL CBE was shown. (b) The levels of oil red O incorporation were quantified by measuring the absorbance of isopropyl alcohol extract at λ = 550 nm. Data were shown relative to AM and expressed as mean ± SD from three independent experiments. ^∗ p < 0.01 versus AM (100%) at same day; °p < 0.05 versus CBE 10 μg/mL at 7 days; ^• p < 0.01 versus CBE 100 μg/mL at 7 days.

Figure 3

Effect of 10 and 100 μg/mL Citrus bergamia extract (CBE) on the lipid content of 14-day differentiated adipocytes stained with oil red O. (a) A representative photograph from three independent experiments of untreated mesenchymal stem cells (Control), 14-day differentiated cells (adipocytes), adipocytes with 10 or 100 μg/mL CBE at 7 and 14 days was shown. (b) The levels of oil red O incorporation was quantified by measuring the absorbance of isopropyl alcohol extract at λ = 550 nm. Data were shown relative to 14-day differentiated adipocytes and expressed as mean ± SD from three independent experiments. ^∗ p < 0.01 versus the 14-day differentiated adipocytes (100%) at same day; °p < 0.01 versus CBE 10 μg/mL at 7 days; ^• p < 0.01 versus CBE 100 μg/mL at 7 days.

Figure 4

Inhibitory effects of 10 or 100 μg/mL Citrus bergamia extract (CBE) on adipocyte differentiation in mesenchymal stem cells induced to differentiate into adipocytes by using adipogenic medium in presence or absence of CBE. At 7, 14, 21, and 28 days, differentiated mesenchymal stem cells adipocytes were examined for PPAR-γ, A-FAB, AMPKα1/2, and p-AMPKα1/2 by Western blot. The levels of proteins were expressed as arbitrary densitometric units (ADU) and the ratio p-AMPKα1/2/AMPKα1/2. Data are shown relative to adipogenic-treated cells and expressed as mean ± SD from three independent experiments. ^∗ p < 0.01 versus the mesenchymal stem cells treated with adipogenic medium at same day; °p < 0.01 versus CBE 10 μg/mL at 28 days; ^• p < 0.01 versus CBE 100 μg/mL at 28 days.

Figure 5

Effects of Citrus bergamia extraction on PPAR-γ, ATGL, HSL, and MGL: 14-days differentiated adipocytes were treated with 10 or 100 μg/mL for 7 or 14 days. The levels of proteins are expressed as arbitrary densitometric unit (ADU). Data were shown relative to 14-day differentiated adipocytes (adipocytes) and expressed as mean ± SD from three independent experiments. ^∗ p < 0.01 versus the 14-day differentiated adipocytes (100%).

Figure 6

Stimulation of lipolysis by 10 or 100 μg/mL Citrus bergamia extract (CBE) in 14-day differentiated adipocytes, assessed by the amount of glycerol released into the media. Data were expressed (μg/mL) as mean ± SD from a representative triplicate experiment. ^∗ p < 0.01 versus the 14-day differentiated adipocytes; °p < 0.05 versus CBE 10 μg/mL at 7 days; ^• p < 0.01 versus CBE 100 μg/mL at 7 days.