Fig 1.
Effect of camphene on lipid profile and metabolism in HepG2 cells-Comparison with well known hypolipidemic compounds.
On day 7, the cells were incubated with the compounds for 24 h in DMEM containing 10% (v/v) human LPDS. HepG2 cells were treated with camphene (lanes: 2–4), mevinolin (lane: 5), atorvastatin (lane: 7), the OSC inhibitor, U18666A (lane: 6) and the ACAT inhibitor, F1394 (lane: 8). Control-non treated cells migrate in places 1 and 9. Camphene was used at concentration of 25, 50 and 100 μM lanes 2, 3 and 4 respectively. Four hours prior to the end of the incubation period, the cells were pulse-labeled with [14C]-acetic acid, sodium salt. This panel shows the autoradiograph of synthesized intracellular lipids separated by TLC after labelling of cells with [14C]-acetate. The positions of the different lipids have been determined using non radioactive standards. The arrows indicate the positions of migration of cholesterol, fatty acids, triglycerides, cholesterol esters, MOS and DOS.
Fig 2.
Effect of camphene, mevinolin and atorvastatin on the incorporation of acetate into de novo synthesized cholesterol.
On day 7, the cells were incubated with the compounds for 24 h in DMEM containing 10% (v/v) human LPDS. Camphene, mevinolin and atorvastatin were received at the concentrations indicated. Four hours prior to the end of the incubation period, the cells were pulse-labeled with [14C]-acetic acid, sodium salt. The incorporation of acetate into cholesterol was determined as described. Results were normalized to the amount of cellular protein and expressed in percent of the respective control incubations. Results are from three independent experiments in triplicates and expressed as the mean ± SD. Significantly different compared to control: p<0.05 (*); p<0.01(**); p<0.001 (***) vs control by Student-Newmann-Keuls test. 25 μM vs 50 μM p<0.05; 25 μM vs 100 μM p<0.01; 50 μM vs 100 μM p>0.05.
Fig 3.
Effect of camphene, mevinolin and atorvastatin on the incorporation of [14C]-acetate into de novo synthesized triglycerides.
On day 7, HepG2 cells were incubated for 24 h with each compound in medium containing 10% (v/v) human LPDS. The cells received camphene, mevinolin and atorvastatin at the concentrations indicated. Four hours prior to the end of the incubation period, the cells were pulse-labeled with [14C]-acetic acid, sodium salt. The incorporation of acetate into triglycerides was determined as described in Materials and Methods section. Results were normalized to the amount of cellular protein and expressed in percent of the respective control incubations. Results are from three independent experiments in triplicates and expressed as the mean ± SD. Significantly different compared to control: p<0.05 (*); p<0.01 (**); p<0.001 (***); p>0.05 and ns (non significant) vs control by Student-Newmann-Keuls test.
Fig 4.
Incorporation of [14C]-acetate and [14C]-oleate in the biosynthetic pathway of triglycerides.
Fig 5.
Effect of camphene on the incorporation of [14C]-acetate into de novo synthesized fatty acids.
On day 7, HepG2 cells were incubated for 24 h with camphene (25, 50 and 100 μM) in medium containing 10% (v/v) human LPDS. Four hours prior to the end of the incubation period, the cells were pulse-labeled with [14C]-acetic acid, sodium salt. The incorporation of acetate into fatty acids was determined as described. Results were normalized to the amount of cellular protein and expressed in percent of the respective control incubations. Results are from three independent experiments in triplicates and expressed as the mean ± SD. Significantly different compared to control: p<0.05 (*); p <0.001 (***) and ns (non significant) vs control by Student-Newmann-Keuls test.
Fig 6.
Effect of camphene on the esterification of fatty acids into triglycerides.
On day 7, HepG2 cells were incubated for 24 h with each compound in medium containing 10% (v/v) human LPDS. The cells received camphene (25, 50 and 100 μM), mevinolin (37 μM) or atorvastatin (10 μM). Four hours prior to the end of the incubation period, the cells were pulse-labeled with [14C]-oleic acid, sodium salt. The incorporation of oleate into triglycerides was determined as described. Results were normalized to the amount of cellular protein and expressed in percent of the respective control incubations. Results are from three independent experiments in triplicates and expressed as the mean ± SD. Results not statistically significant vs control, ns (non significant) by Student-Newmann-Keuls test.
Fig 7.
Effect of camphene on the protein expression of apoAI.
Panel A. On day 7, HepG2 cells were incubated for 48 h with camphene (1, 5, 10, 37 and 50 μM) in DMEM containing 10% LPDS. Total cell protein was extracted and 50 μg of protein were subjected to SDS PAGE electrophoresis and analyzed by Western immunoblotting using goat anti-apoAI antibody as described. Mouse β-actin was used to control for equal loading and normalization. Relative intensity of the bands was quantified using Phosphorimager. Values express the apoAI/actin ratio and are calculated by comparison to control samples the value of which is defined as 1. Values are means ± SD of three independent experiments in triplicates. p<0.05 (*); p<0.001 (***); p>0.05 and ns (non significant) vs control by the Student-Newmann-Keuls test. 1 μM is significantly different from 37 and 50 μM with p<0.01 and p<0.001 respectively. 5 μM treatment is significantly different from 37 and 50 μM with p<0.05 and p<0.001 respectively. 10 μM treatment is significantly different from 37 and 50 μM treatments with p<0.05 and p<0.001 respectively. Camphene concentration of 37 μM is significantly different from 50 μM with p<0.05. Panel B. A representative Western blot is shown.
Fig 8.
Effect of camphene and mevinolin on the protein expression of apoAI.
Panel A. On day 7, HepG2 cells were incubated for 48 h with camphene (37 μM) and mevinolin (37 μM) in DMEM containing 10% LPDS. Total cell protein was extracted and 50 μg of protein were separated by SDS PAGE and analyzed by Western immunoblotting using goat anti-apoAI antibody as described. Mouse β-actin was used to control for equal loading and normalization. Relative intensity of the bands was quantified using Phosphorimager. Values express the apoAI/actin ratio and are calculated by comparison to control samples the value of which is defined as 1.Values are means ± SD of three independent experiments in triplicates. p<0.001 (***) and ns (non significant) vs control by the Student-Newmann-Keuls test. Panel B. A representative Western blot is shown.
Fig 9.
Effect of camphene and mevinolin on the protein expression of SREBP-1. A and B panels.
Effects of the compounds on the mature and the precursor form of SREBP-1 respectively. On day 7, HepG2 cells were incubated for 24 h with camphene (37 μM) and mevinolin (37 μM) in DMEM containing 10% LPDS. 25 μg/ml ALLN were added for the last 5 h before harvesting, in order to stabilize the short-lived nuclear forms. Total cell protein was extracted and 50 μg of protein were separated on a 8% polyacrylamide gel and analyzed by Western immunoblotting using rabbit anti-SREBP-1 as described. Mouse β-actin was used to control for equal loading and normalization. Relative intensity of the bands was quantified by Phosphorimager. Values express the SREBP-1/actin ratio and are calculated by comparison to control samples the value of which is defined as 1.Values are means ± SD of three independent experiments in triplicates. p<0.05 (*); p<0.001 (***); p>0.05 and ns (non significant) vs control by the Student-Newmann-Keuls test. Panel C. A representative Western blot is shown. The upper band is SREBP-1 precursor (125 kDa) and the lower band is mature SREBP-1 (68 kDa).
Fig 10.
Effect of camphene and mevinolin on the protein expression of the mature form of SREBP-2.
Panel A. On day 7, HepG2 cells were incubated for 24 h with camphene (37 μM) and mevinolin (37 μM) in DMEM containing 10% LPDS. 25 μg/ml ALLN were added for the last 5 h before harvesting, in order to stabilize the short-lived nuclear forms. Total cell protein was extracted and 50 μg of protein were separated on a 8% polyacrylamide gel and analyzed by Western immunoblotting using rabbit anti-SREBP-2 as described. Mouse β-actin was used to control for equal loading and normalization. Relative intensity of the bands was quantified by Phosphorimager. Values express the SREBP-2/actin ratio and are calculated by comparison to control samples the value of which is defined as 1.Values are means ± SD of three independent experiments in triplicates. p<0.05 (*); p>0.05 and ns (non significant) vs control by the Student-Newmann-Keuls test. Panel B. A representative Western blot is shown. SREBP-2 mature form is about 70 kDa.
Fig 11.
Effect of camphene on MTP expression.
On day 7, HepG2 cells were incubated with indicated concentrations of camphene for 24 h in DMEM containing 10% (v/v) human LPDS. Panel A. Total cell protein was extracted and 50 μg of protein were separated on a 12% polyacrylamide gel and analyzed by Western immunoblotting using goat anti-MTP as described in Materials and Methods section. Mouse β-actin was used to control for equal loading and normalization. Relative intensity of the bands was quantified by Phosphorimager. Values express the MTP/actin ratio and are calculated by comparison to control samples the value of which is defined as 1. Panel B. A representative Western blot is shown. Panel C. Total RNA was extracted and subjected to Real Time PCR. The results represent the relative MTP mRNA normalized to endogenous YWHAZ mRNA. The same results were observed after normalization to the housekeeping gene actin (data not shown). The abundance of mRNA in the control-untreated cells was set at 1. Data represent means ± SD of three independent experiments. p<0.001 (***) and p<0.01 (**) vs control cells by the Student-Newmann-Keuls test.