Fig. 7

Density distribution of secreted apoB-Lp in control and taxifolin-treated cells. Treated and untreated HepG2 cells were grown, pulsed, and chased as described in Fig. 5. The extracellular fraction was collected at the end of the chase period (2 h) and subjected to sucrose gradient ultracentrifugation. After centrifugation, gradient fractions were collected and immunoprecipitated with an anti-apoB antibody. The immunoprecipitates were analyzed by SDS-PAGE and fluorography, and apoB radioactivity was quantified by cutting and scintillation counting of the apoB band. Data represent means ± SD of two independent experiments performed in duplicate.

Benoist, Nicodeme, and Grand-Perret (21) have suggested that another protease, a DTT-sensitive protease, may be involved in the early stages of apoB degradation. Hence, we examined the possibility that such a protease could mediate the effect of taxifolin on apoB secretion. We used a DTT protocol as described by Shelness and Thornburg (22). Taxifolin-treated and untreated cells were first pretreated for 1 min with and without DTT (2 mM) in the presence and absence of taxifolin. After a 10-min pulse, the cells were chased for 5 min and harvested for apoB immunoprecipitation. Interestingly, as shown in Fig. 8, addition of DTT to the taxifolin-treated cells (lanes 7 and 8) increased apoB cellular content comparable to the untreated DTT control cells (lanes 5 and 6). Despite a reduction in protein synthesis with DTT as has been observed by others (21, 22), the effect of taxifolin on early apoB degradation was virtually prevented. This suggest that a putative DTT-sensitive protease may be involved.

Taxifolin partially inhibits the stimulatory effect of oleate and 25-hydroxycholesterol on apoB secretion

To investigate the effect of taxifolin on apoB secretion under lipid-rich conditions, both oleate (360 μM complexed to BSA) and 25-hydroxycholesterol (10 μg/ml dissolved in ethanol) were added overnight in the presence or absence of taxifolin (200 μM). Figures 9A and B demonstrate the amount of apoB secreted over a 2-h chase in the presence and absence of taxifolin. Experiments with oleate were conducted in the presence of medium plus BSA, medium plus BSA/taxifolin, medium plus BSA/oleate, and medium plus BSA/oleate/taxifolin. Treatment with oleate showed a 4-fold stimulation of apoB secretion compared with BSA-untreated cells. Coincubation with both oleate and taxifolin resulted in a lower stimulation of apoB secretion (39% decrease vs. oleate-treated cells; reproduced in one other independent experiment). Similarly, 25-hydroxycholesterol also resulted in a significant elevation of apoB secretion when compared with the ethanol-treated control cells (2-fold stimulation). In contrast, the effect of 25-hydroxycholesterol with taxifolin was more pronounced than with oleate, as taxifolin was shown to markedly reverse the stimulatory effect of 25-hydroxycholesterol on apoB secretion (75% vs. 25-hydroxycholesterol-treated cells; reproduced in one other independent experiment).

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