Fig. 3

Effects of taxifolin on HMGR and ACAT activity. HepG2 cells were treated with and without 200 μM taxifolin for a total of 24 h. HMGR activity was assayed in permeabilized cells in the presence and absence of taxifolin by [¹⁴C]HMG-CoA labeling. The mevalonolactone formed was extracted, separated by TLC, and quantitated by scintillation counting. ACAT activity was determined in situ by [¹⁴C]oleic acid labeling. The CE formed was extracted and separated by TLC, and the formation of CE was measured by scintillation counting. The results are expressed as a percentage of control (set as 100%). Values represent the mean ± SD of three independent experiments in duplicate. ^* P < 0.05 versus control.

Taxifolin also inhibits the synthesis and secretion of TAG and phospholipids

To evaluate further the effects of taxifolin on the synthesis and secretion of TAG and phospholipids, HepG2 cells were labeled with [¹⁴C]glycerol with and without 200 μM taxifolin for a total of 24 h. As summarized in Fig. 4, taxifolin showed an inhibitory effect on the accumulation of TAG (59 ± 1%, P < 0.05, n = 3) and phospholipids (15 ± 2%, P < 0.05, n = 3) in the cell. Similar inhibitory effects on the secretion of TAG (68 ± 3%, P < 0.05, n = 3), with a more pronounced inhibition of phospholipid secretion (57 ± 1%, P < 0.05, n = 3), were also observed when compared with their synthesis level.

Taxifolin inhibits apoB and stimulates apoA-I secretion

A metabolic pulse-chase labeling experiment was performed to assess the fate of nascent apoB and apoA-I in untreated control and taxifolin-treated cell cultures. Cells were pulsed with [³⁵S]methionine/cysteine for 10 min, and chased with excess cold methionine/cysteine medium ± taxifolin for up to 120 or 180 min. Aliquots of the intracellular and extracellular fractions were collected at various time points and analyzed. A preliminary experiment showed a 10- and 20-min delay in reaching peak incorporation of [³⁵S]methionine/cysteine into full-length apoA-I and apoB, respectively (data not shown).

Figure 5A is a fluorograph showing the amount of apoB synthesized and secreted by HepG2 cells in the presence and absence of taxifolin in a typical pulse-chase experiment (reproduced in two other independent experiments; results pooled in Fig. 6). A 64% decrease in the incorporation of [³⁵S]methionine/cysteine into immunoprecipitable apoB was apparent with taxifolin at the 20-min chase time (representing peak incorporation). When the radioactivity was further chased, a gradual reduction in the intracellular labeled apoB was noted with a simultaneous increase in secreted labeled apoB. In the presence of taxifolin, a significant reduction in apoB secretion was noted (70%) when compared with the untreated control cells at the 180-min chase time (representing peak secretion). This effect on secretion with taxifolin was comparable to that of synthesis, indicating that the reduction in apoB secretion may be due to a decrease in newly synthesized apoB, possibly through a transcriptional and/or translational mechanism(s).

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