Fig. 9

Effect of taxifolin on apoB secretion in oleate- and 25-hydroxycholesterol-treated cells. HepG2 cells were treated with 200 μM taxifolin for 24 h in the presence or absence of BSA-bound oleate (360 μM) or 25-hydroxycholesterol (10 μg/ml). Cells were pulsed and chased for 2 h. Medium was collected and apoB was immunoprecipitated, followed by SDS-PAGE and fluorography. Quantification was done by scintillation counting of the apoB band. (A) Representative fluorograph showing the amount of radiolabeled apoB secreted from cells treated with medium plus BSA, medium plus BSA/taxifolin, medium plus BSA/oleate, and medium plus BSA/oleate/taxifolin. (B) Representative fluorograph showing the amount of radiolabeled apoB secreted from cells treated with medium, medium plus taxifolin, medium plus 25-hydroxycholesterol, and medium plus 25-hydroxycholesterol/taxifolin. Data are typical of an experiment performed twice in duplicate.

The present study provides evidence that taxifolin is capable of influencing lipid and apolipoprotein production. Initial studies examined the effects of taxifolin on lipid synthesis and secretion in HepG2 cells. Our results confirmed that taxifolin is able to decrease [¹⁴C]acetate incorporation into cholesterol in a dose- and time-dependent manner. Interestingly, acetate incorporation was inhibited by as much as 86% at 200 μM, a concentration at which cell viability was not altered. Consequently, this concentration was chosen in the following experiments and represents a pharmacological dose. Under this condition, taxifolin was shown to equally inhibit both free cholesterol and CE synthesis. Moreover, using [¹⁴C]glycerol incorporation studies, TAG and phospholipid synthesis was also found to be significantly reduced. These results with lipids are, on one hand, consistent with that from silybin and silymarin. Studies by Nassuato et al. (9), Montanini et al. (23), and Petronelli et al. (24) showed that silybin and silymarin decreased hepatic synthesis of cholesterol, phospholipids, and TAG. On the other hand, our results are in contrast with the citrus flavonoids hesperetin and naringenin, which were shown to have little inhibitory effect and, in some cases, a stimulatory effect on free cholesterol, TAG, and phospholipid synthesis in HepG2 cells (7, 8). The discrepancy in results may stem from structural differences in the flavonoid molecules. Accordingly, taxifolin appears to be more effective than hesperetin and naringenin in reducing lipid synthesis. It is possible that taxifolin may have a more global transcriptional regulation in lipogenesis with such proteins as the sterol regulatory element-binding protein. Secretion of lipids into the culture medium was also investigated in taxifolin-treated cells. Our results indicated a similar decrease in free cholesterol, CE, and TAG, with a more pronounced inhibition of phospholipid secretion compared with the effect observed on phospholipid synthesis.

In elucidating the mechanism of action on cholesterol synthesis, we examined the effect of taxifolin on HMGR activity, a key enzyme in cholesterol biosynthesis. Nassuato et al. (9) demonstrated a dose-dependent inhibition of HMGR by silybin. Our results showed that the taxifolin moiety of silybin also inhibited HMGR activity. Although the level at which taxifolin may be exerting its effect on HMGR gene expression remained unknown, the data did yield information about the level at which taxifolin influences hepatic lipid synthesis. Thus, taxifolin is suggested to behave like a statin drug. Unlike these common cholesterol-lowering drugs, taxifolin may be hepatoprotective and exhibit beneficial antioxidant characteristics (25, 26).

Evidence that flavonoids may also inhibit ACAT activity and cholesterol esterification has been reported by a number of studies (7, 10, 27, 28). Hence, we examined whether taxifolin shares this property. We have shown that taxifolin indeed reduced the incorporation of [¹⁴C]oleate into cellular CE in situ, suggesting ACAT activity may also be involved. However, as previously noted, we cannot completely rule in ACAT activity because the decreased HMGR activity may have led to the decreased esterified cholesterol due to the lower levels of substrate.

Because apolipoproteins play an important role in cholesterol transport, we continued our study by investigating the effects of taxifolin on apoA-I and apoB synthesis and secretion. Although apoA-I is associated mainly with HDL and is involved in the reverse cholesterol transport pathway, apoB is associated with LDL and is said to be atherogenic because it delivers cholesterol to the cell. Using pulse-chase experiments, the rates of synthesis and secretion were estimated. We found the incorporation of [³⁵S]methionine/cysteine to be markedly decreased in cellular apoB and increased in cellular apoA-I, suggesting that changes in the rate of apolipoprotein synthesis may be involved. The changes in the amount of cellular apoA-I and apoB in the presence of taxifolin translated into a similar effect on apoA-I and apoB levels appearing in the medium. Because synthesis was found to be affected, the possibility that taxifolin altered transcription and/or translation of apoB and apoA-I was not excluded. However, in the case of apoB, these mechanisms of action are unlikely because most reports have indicated that changes in apoB secretion are primarily regulated co- and posttranslationally through intracellular degradation [reviewed in ref. (19)]. According to this model, newly synthesized apoB associated with the cytosolic side of the ER membrane is degraded cotranslationally by the proteasome (20), whereas fully translocated apoB is thought to be degraded either posttranslationally by an ER-localized cysteine protease (29) or translocated back out to the cytosol cotranslationally for proteasomal degradation (30). In addition, there is indication that a DTT-sensitive protease may be involved in the early stages of apoB degradation (21, 31). Our results are consistent with early degradation represented by a significant decrease in the intracellular concentration of newly synthesized apoB at the beginning of the chase in our metabolic pulse-chase labeling experiments. Although the apoB mRNA level was found to remain essentially unchanged, evidence of cotranslational control was provided by our protease data. Interestingly, our data support the hypothesis that taxifolin is involved at an early stage of apoB processing and that DTT can reverse the effect of taxifolin on apoB secretion. In contrast, the lack of response to the proteasome inhibitor ALLN argues against involvement of the proteasome in taxifolin-induced changes in apoB secretion. Taken together, the mechanism of taxifolin-mediated lipoprotein assembly with apoB appears to involve a DTT-sensitive, ALLN-insensitive degradation pathway. These results are compatible with Benoist, Nicodeme, and Grand-Perret (21), whose microsomal triglyceride transfer protein inhibitor was shown to act rapidly through a DTT-sensitive, ALLN-insensitive proteolytic pathway. Also, this is compatible with the results from Borradaile, Carroll, and Kurowska (8) on the inhibitory effects of naringenin and hesperetin on apoB secretion. As observed by these investigators, coincubation with the proteasome inhibitor MG132 failed to increase apoB secretion, indicating that proteasomal degradation was not involved. The exact nature and role of our DTT-sensitive protease in apoB degradation remain to be elucidated.

The finding of reduced apoB secretion of cells incubated with taxifolin is also supported by the analysis of the lipoprotein fraction distribution, which showed a reduced accumulation of labeled LDL-like apoB in the medium of cells incubated with taxifolin. No major shift in the distribution of secreted lipoproteins was observed when compared with untreated control cells. Together, these results indicated that taxifolin decreased the number of apoB-Lp secreted without a major change in the density of the assembled lipoproteins.

The effect on apoB synthesis and secretion was found to be specific when compared with apoA-I and albumin. Both albumin and apoA-I synthesis and secretion were shown to be stimulated. This effect is interesting and may reflect a global transcriptional effect. This is compatible with the mechanism of action of silybin, the flavolignan containing a taxifolin moiety, which can influence RNA polymerase activity thought to be important in the repair phase of liver damage (18). However, the apoA-I transcription rate remained essentially unchanged with taxifolin, suggesting that other mechanism(s) may be involved. Nevertheless, silybin, as well as silymarin, were shown in hypercholesterolemic rats to increase HDL-cholesterol levels (11), which is compatible with our findings on apoA-I. Thus, taxifolin appears to have benefits related to HDL metabolism.

The assembly of apoB-Lp particles is a complex process that requires the coordinated synthesis and assembly of apoB, TAG, CE, phospholipids, and other components. Because lipid availability is a major determining factor in the assembly and secretion of apoB-Lp, we studied the effects of exogenous lipids on the ability of taxifolin to reduce apoB secretion in HepG2 cells. The addition of oleate and 25-hydroxycholesterol has been previously shown to stimulate the secretion of apoB in these cells (32, 33). Oleate presumably functions to enhance TAG synthesis, whereas 25-hydroxycholesterol increases CE content. Results of these experiments showed a remarkable increase in apoB secretion in oleate- and 25-hydroxycholesterol-treated cells. On addition of taxifolin with oleate, a moderate reduction in apoB secretion was observed. Interestingly, however, the effects were more pronounced with 25-hydroxycholesterol, which markedly diminished the secretion of apoB. The fact that taxifolin reversed the effect of each lipid differently is intriguing and may reflect that CE content, rather than TAG, is primarily associated with changes in apoB secretion under taxifolin treatment. Thus, taxifolin may be exerting its apoB-Lp-lowering action by limiting CE availability essential for the assembly of apoB-Lp.

In summary, the data in this report indicate that taxifolin inhibited the synthesis and secretion of a number of lipids, in addition to decreasing apoB and increasing apoA-I secretion. This supports the theory that taxifolin, the moiety of the flavolignan silybin, may represent a potentially important method of controlling atherogenesis. This may also account for the superior hypocholesterolemic activity of silymarin relative to silybin. The suggested statin-like activity of taxifolin may lead to an alternative neutraceutical agent with combined hypocholesterolemic and antioxidant properties.

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Acknowledgments

The investigation was supported by a Research Centers of Minority Institutions (RCMI) award, P20 RR/AI 11091, from the National Center for Research Resources, National Institutes of Health.

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Footnotes

· ↵1 Part of this work was presented at the American Heart Association 72nd Scientific Sessions, Atlanta, GA, in November 1999, and printed in abstract form in Circulation. 1999. 100: I-109.

· Abbreviations:

ACAT

acyl-CoA:cholesterol acyltransferase

ALLN

N -acetyl-leucyl-leucyl-norleucinal

apoA-I

apolipoprotein A-I

apoB

apolipoprotein B-100

apoB-Lp

apoB-containing lipoprotein

ATCC

American Type Culture Collection

BSA

bovine serum albumin

CE

cholesteryl ester

CVD

cardiovascular disease

DTT

dithiothreitol

EBSS

Earle's balanced salt solution

EDTA

ethylenediaminetetraacetic acid

ER

endoplasmic reticulum

HDL

high density lipoprotein

HMGR

3-hydroxy-3-methylglutaryl-coenzyme A reductase

LDH

lactate dehydrogenase

LDL

low density lipoprotein

SDS-PAGE

sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SF-RPMI

serum-free RPMI

TAG

triacylglycerol

TCA

trichloroacetic acid

TLC

thin-layer chromatography.

· Received May 18, 2000.

· Revision received July 24, 2000.

· Copyright © 2000 by Lipid Research, Inc.

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