Inhibitory activity of diacylglycerol acyltransferase (DGAT) and microsomal triglyceride transfer protein (MTP) by the flavonoid, taxifolin, in HepG2 cells: potential role in the regulation of apolipoprotein B secretion

Atherosclerosis 176 (2004) 247–253

Inhibitory activity of diacylglycerol acyltransferase (DGAT) and microsomal triglyceride transfer protein (MTP) by the flavonoid, taxifolin, in HepG2 cells: potential role in the regulation of apolipoprotein B secretion Adele Casaschi, Brent K. Rubio, Geoffrey K. Maiyoh, Andre G. Theriault∗ Division of Medical Technology, John A. Burns School of Medicine, University of Hawaii at Manoa, 1960 East-West Road, Biomed C-206, Honolulu, HI 96822, USA Received 14 October 2003; received in revised form 2 March 2004; accepted 17 May 2004 Available online 28 July 2004

Abstract The purpose of the present study was to examine the role of taxifolin, a plant flavonoid, on several aspects involving apolipoprotein B (apoB) secretion and triglyceride (TG) availability in HepG2 cells. Taxifolin was shown by ELISA to markedly reduce apoB secretion under basal and lipid-rich conditions up to 63% at 200 ␮mol/L. As to the mechanism underlying this effect, we examined whether taxifolin exerted its effect by limiting TG availability in the microsomal lumen essential for lipoprotein assembly. Taxifolin was shown to inhibit microsomal TG synthesis by 37% and its subsequent transfer into the lumen (−26%). The reduction in synthesis was due to a decrease in diacylglycerol acyltransferase (DGAT) activity (−35%). The effect on DGAT activity was found to be non-competitive and non-transcriptional in nature. Both DGAT-1 and DGAT-2 mRNA expression remained essentially unchanged suggesting the point of regulation may be at the post-transcriptional level. Evidence is accumulating that microsomal triglyceride transfer protein (MTP) is also involved in determining the amount of lumenal TG available for lipoprotein assembly and secretion. Taxifolin was shown to inhibit this enzyme by 41%. Whether the reduction in TG accumulation in the microsomal lumen is predominantly due to DGAT and/or MTP activity remains to be addressed. In summary, taxifolin reduced apoB secretion by limiting TG availability via DGAT and MTP activity. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Bioflavonoid; Apolipoprotein B; Triglyceride; Diacylglycerol acyltransferase; Microsomal triglyceride transfer protein

1. Introduction Apolipoprotein B-100 (apoB) is a large amphipathic protein that provides the framework for the assembly of very low density lipoprotein (VLDL) [1]. Attempt to lower the production of apoB is thought to reduce the risk of de-

Abbreviations: apoB, apolipoprotein B; apoB-Lp, apoB-containing lipoprotein; ACAT, acyl CoA:cholesterol acytltransferase; BSA, bovine serum albumin; CE, cholesterol ester; DGAT, diacylglycerol acyltransferase; ELISA, enzyme-linked immunosorbent assay; FBS, fetal bovine serum; HMG, hydroxymethylglutaryl; LDL, low density lipoprotein; MTP, microsomal triglyceride transfer protein; TG, triglyceride; TCA, trichloroacetic acid; TLC, thin layer chromatography; VLDL, very low density lipoprotein ∗ Corresponding author. Tel.: +1 808 956 8656; fax: +1 808 956 5457. E-mail address: [email protected] (A.G. Theriault).

veloping coronary artery disease (CAD). In recent years, several flavonoids have been shown to reduce apoB production in cell culture [2–6]. A number of lipogenic enzymes have been shown to be involved, including hydroxymethylglutaryl (HMG) CoA reductase, acyl CoA: cholesterol acyltransferase (ACAT), microsomal triglyceride transfer protein (MTP), and recently diacylglycerol acyltransferase (DGAT) [3,4,7]. However, the enzyme(s) and lipid(s) predominantly responsible in the assembly of apoB-containing lipoprotein (apoB-Lp) by the flavonoids have not been thoroughly investigated. Borradaile et al. [8] recently made progress in this area by ruling out ACAT activity and cholesterol ester (CE) availability in the regulation of apoB-Lp by the citrus flavonoid, naringenin, in HepG2 cells. Using specific ACAT, HMG CoA reductase, and MTP inhibitors, they concluded that naringenin inhibited apoB secretion

0021-9150/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.atherosclerosis.2004.05.020

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HepG2 cells as the model system. Milk thistle is a commercially available herbal supplement which has long been used for its hepatoprotectant and antioxidant properties [13]. This study could contribute to a better understanding of this herbal extract in the treatment of hypertriglyceridemia.

2. Materials and methods Fig. 1. Structure of taxifolin

by limiting triglyceride (TG) availability in the active endoplasmic reticulum (ER) lumen necessary for lipoprotein assembly [8,9]. While cytosolic TG content was shown to increase slightly and microsomal membrane TG synthesis remained essentially unchanged, the critical ER lumenal TG pool decreased significantly. The authors accounted the decrease in lumenal TG content to MTP inhibition. MTP, in addition to catalyzing the transfer of lipids to nascent apoB molecules, has been shown to facilitate the accumulation and attainment of TG within the ER lumen [10]. Insufficient MTP activity was thereby suggested to be the primary reason for the lack of transfer of newly synthesized TG from the microsomal membrane to the active lumenal pool under naringenin treatment [9]. Although, their conclusion does agree with their findings, we have observed discrepancies on the effects of flavonoids on TG synthesis. While the studies on naringenin showed no or slightly increased effects on TG synthesis [8,9], our flavonoid, taxifolin (Fig. 1), was shown to decrease TG synthesis [3]. The cause for the discrepancy is unknown, but may suggests that not all flavonoids behave similarly on lipid metabolism. We sought to examine the effects of taxifolin on TG synthesis more closely and the possibility that lack of substrate (i.e., TG) may represent an alternative mechanism for insufficient transfer of newly synthesized TG from the microsomal membrane to the active lumenal pool. We have recently showed that the flavonoid, quercetin, inhibited TG synthesis, in part, via DGAT activity, using the intestinal cell-line, CaCo-2 [4]. DGAT is a key microsomal enzyme in TG biosynthesis which acylates diacylglycerol at the sn-3 position, using fatty acyl CoAs resulting in the formation of TG. The enzyme is involved in both the glycerol–phosphate and monoacylglycerol pathways. It is considered to be a key step in TG synthesis (reviewed in [11]). While the study on CaCo-2 cells was crucial in identifying flavonoids as a potential DGAT inhibitor, the mechanism by which flavonoids affected DGAT activity and the contribution of DGAT inhibition on TG synthesis and apoB secretion in hepatocytes have never been investigated. Since TG availability is considered a major contributor in the regulation of hepatic apoB secretion [12], the purpose of the present study was to examine the role of taxifolin, a plant flavonoid derived from the seed of milk thistle (Silybum marianumL.), on several aspects involving apoB secretion and TG availability, using

2.1. Cell Culture Monolayer HepG2 cell cultures (HB 8065; American Type Culture Collection, Rockville, MD, USA) were maintained in RPMI-1640 medium with 10% fetal bovine serum (FBS) (InVitrogen Life Technologies Corp., Carlsbad, CA, USA) at 37 ◦ C with 5% CO2 and subcultured in 35-mm diameter dishes (Corning Costar Corp., Cambridge, MA, USA) to about 80% confluent. In studies with oleate, a serum-free (SF) medium containing bovine serum albumin (BSA) complexed to oleic acid (OA) (0.81 mmol/L OA: 0.1 mmol/L BSA) was used. In experiments without oleate, cells were treated with taxifolin in SF media. Taxifolin (approximately 95% pure) was purchased from Sigma Chemicals Co. (St. Louis, MO, USA). The compound was dissolved in DMSO. An appropriate amount of stock solution was diluted in culture medium to give a final DMSO concentration of no greater than 0.25%. The stock solution was kept at 4 ◦ C for no longer than 4 weeks. Untreated control cells were treated with the solvent (DMSO) only. All treatments were for 24 h. 2.2. Apolipoprotein B ELISA ApoB secreted into the medium was determined using a non-competitive binding enzyme-linked immunosorbent assay (ELISA) essentially as described [14]. Spectrophotometric readings were made, using a microplate reader (Thermomax, Molecular Devices, Sunnyvale, CA, USA). Intra- and inter-assay coefficients of variation were 4.5 and 7.5%. Cell proteins were digested in 1 mL of 0.1N NaOH and measured as described below. 2.3. Microsomal triglyceride synthesis To measure the rate in microsomal TG synthesis, treated and untreated cells from 100-mm dishes were labeled with 10 ␮Ci/mL [2-3 H]glycerol (200 mCi/mmol, Perkin–Elmer Life Science Research Products, Boston, MA, USA) for 2 h and fractionated into cytosol, microsomal membrane, and microsomal lumen. The fractionation procedure was performed essentially as described [8]. After labeling, cells were scraped with 1.5 mL of a Tris-sucrose buffer (10 mM Tris–HCl, 250 mM sucrose, pH 7.4) and homogenized by 15 strokes using a Potter–Elvehjem homogenizer. The post-nuclear supernatant was obtained by centrifugation for 10 min at 10,000 × g. Subsequently, the microsomes

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and cytosol were separated, using a Beckman SW-55 rotor (100,000 × g, 4 ◦ C, 1 h). The total microsomes (consisting of ER and Golgi) were rinsed twice with TRIS-sucrose buffer and the lumenal content was released with 0.1 mM sodium carbonate (pH 11.3). The lumenal content was separated from the microsomal membrane by ultracentrifugation (100,000 × g, 4 ◦ C, 1 h). Lipids in each fraction were extracted with hexane-isopropanol 3:2 (v/v) and subjected to thin layer chromatography (TLC) as described by Theriault et al. [3]. Radioactivity associated with TG was counted by liquid scintillation counting and normalized to cell protein. Cell protein in parallel dishes were digested in 1 mL of 0.1N NaOH and measured as described below. 2.4. DGAT activity assay Esterification of diacylglycerol into TG was measured by using [palmitoyl 1-14 C]palmitoyl-CoA (40–60 mCi/mmol (Perkin–Elmer) essentially as described by Grigor and Bell [15]. The TG formed was extracted from the assay reaction tube and subjected to TLC in chloroform-acetic acid (96:4). Preliminary experiments indicated whole cell DGAT activity correlated with microsomal DGAT activity and the assay was linear up to 1000 ␮g/mL of cell protein (data not shown). In some experiments, the direct effect of taxifolin on DGAT activity was determined by adding the agent directly to the assay reaction containing control HepG2 microsomes (0.6 ␮g microsomal protein/mL). 2.5. Measurement of DGAT mRNA abundance Relative RT-PCR was performed in order to determine the levels of DGAT-1 and DGAT-2 gene expression. Isolated total RNA was reversed transcribed using 100 U/␮L SuperSCRIPTII RNase H− reverse transcriptase (Invitrogen) primed with 0.3 ␮g/␮L random primers (Invitrogen). DGAT mRNA was amplified in a Eppendorf Mastercycler (Eppendorf Scientific Inc., Westbury, NY, USA) using 10 pmol each of DGAT 1-specific primers (sense 5 -GGCCTTCTTCCACGAGTACC-3 ; antisense 5 -GGCCTCATAGTTGAGCACG-3 ) or DGAT2-specific primers (sense 5 -CTCAGACCATAGCCTAAACC-3 ; antisense 5 -CAGCTTAGGGGTGTGACATC-3 ), and Taq DNA polymerase (Eppendorf Scientific) according to the following thermal cycle: 30 s at 94 ◦ C, 30 s at 55 ◦ C, and 30 s at 72 ◦ C for 31 (DGAT-1) or 34 (DGAT-2) cycles (determined to lie within the linear amplification phase). 18S rRNA was used as an internal standard and was co-amplified, using primers and competimers from Ambion Inc. (Austin, TX), using a 1:9 primer:competimer ratio. Amplified products (216 bp for DGAT-1, 307 bp for DGAT-2, and 488bp for 18S rRNA) were run on a 2% agarose gels and visualized by staining with ethidium bromide. Band intensities were analyzed densitometrically using the Gel DocTM Gel Documentation System (Bio-Rad Laboratories Inc., Hercules, CA, USA)

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and DGAT mRNA levels were normalized with respect to 18S rRNA. 2.6. Other methods Cell protein content was measured according to Bradford [16] (i.e., Bio-Rad), using BSA as the standard. Albumin secreted into the medium was performed using an ELISA kit from Bethyl Laboratories Inc. (Montgomery, TX, USA). Media was diluted 1:300 in PBS and added into each well. MTP activity in cell extracts was measured by a fluorescent assay according to the manufacturer’s protocol (Roar Biomedical, New York, NY, USA). The activity of lactate dehydrogenase (LDH) released into the media was measured spectrophotometrically using the CytoTox 96 non-radioactive cytotoxicity assay according to the manufacturer’s protocol (Promega Corp., Madison, WI). Cell viability assay was determined by the trypan blue exclusion technique according to the protocol provided by Invitrogen. Viable and non-viable cells were counted, using a neuenbower hemocytometer. 2.7. Statistical analysis Statistical differences were analyzed by using Student’s t-test with the level of significance set at 0.05.

3. Results 3.1. Taxifolin inhibits apoB secretion under basal and lipid-rich conditions Initial studies were performed to determine an optimal concentration of taxifolin that would reduce apoB secretion in a specific manner. We analyzed the effects of taxifolin on apoB and albumin secretion under SF condition, using an ELISA method. As shown in Fig. 2, taxifolin reduced apoB secretion in a dose-dependent manner after 24-h incubation. Percent inhibition was 24 ± 10% at 75 ␮mol/L, 31 ± 5% at 100 ␮mol/L, and 63 ± 4% at 200 ␮mol/L (versus untreated control; n = 3). As a control, albumin secretion was also measured. The secretion of albumin was essentially unaffected by taxifolin over the range of dosages tested (+1 ± 10% at 75 ␮mol/L, +1 ± 3% at 100 ␮mol/L, and + 12 ± 5% at 200 ␮mol/L versus untreated control), indicating the effects on apoB secretion to be specific. Cell viability at the 200 ␮M concentration, as assessed by the trypan blue exclusion technique and leakage of LDH into the media, was not compromised (data not shown). Consequently, we chose the maximum, non-toxic concentration of 200 ␮mol/L in the following experiments. This concentration represented a pharmacological dose. Since, HepG2 cells are known to be highly dependent on a high concentration of exogenous fatty acids to maintain an adequate supply of lipids for lipoprotein assembly [17], we

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Fig. 2. Effects of taxifolin on apolipoprotein B (apoB) and albumin secretion. HepG2 cells were treated with various concentrations of taxifolin (75–200 ␮mol/L) for 24 h in SF media. Media was collected and apoB/albumin secretion was measured by ELISA. Data are expressed as a percentage of control (set as 100%). Values represent mean ± S.D. of three independent experiments performed in duplicate. ApoB mass in control cells was 52 ± 4 ␮g/mg cell protein (exp. 1), 50 ± 7 ␮g/mg cell protein (exp. 2), and 46 ± 6 ␮g/mg cell protein (exp. 3).

cells [12]. Cells were treated for 22 h with or without taxifolin (200 ␮mol/L) in the presence of oleate (0.81 mmol/L) and labeled with [3 H]glycerol under the same condition for an additional 2 h. The incorporation of [3 H]glycerol into TG from the isolated microsomal membrane was analyzed by TLC and scintillation counting. We observed a significant decrease in microsomal TG synthesis under taxifolin treatment (Table 1). Percent inhibition was 37% (n = 3, P < 0.05 versus untreated control). These results compared well with our previous finding in which taxifolin (200 ␮mol/L, 24 h, under SF condition) inhibited total cellular TG synthesis by 59% [3]. We further examined if the decrease in TG (i.e. substrate) in the microsomal membrane may also limit its accumulation into the lumen. It is the lumenal TG pool which has the ability to regulate apoB secretion in HepG2 cells [17]. We indeed observed a reduction in the influx of membrane TG into the lumen. The accumulation of newly synthesized TG into the lumen decreased by 26% (n = 3, P < 0.05 versus untreated control). This reduction was also coupled with a reduction in TG accumulation in the cytosol (39%, n = 3, P < 0.05 versus untreated control). 3.3. Taxifolin decreases cellular DGAT activity

investigated the effect of taxifolin on apoB secretion under oleate treatment. Oleate (0.81 mmol/L) complexed to BSA (0.1 mmol/L) was added in the presence and absence of taxifolin (200 ␮mol/L). In comparison to the untreated control, we observed a significant reduction in apoB secretion under taxifolin treatment. Percent inhibition was 33 ± 8% (n = 3, P < 0.05 versus BSA control) (data not shown). The reduction was not, however, as great as under basal conditions. The mechanism by which taxifolin inhibited apoB secretion was previously shown by pulse-chase experiments to be the result of enhanced intracellular protein degradation [3].

To address the mechanism for the reduction in TG synthesis, the activity of DGAT was measured. Cell homogenates were first prepared from cells treated with or without taxifolin (200 ␮mol/L) in the presence of oleate (0.81 mmol/L) for 24 h. DGAT activity was then determined by the incorporation of [14 C]palmitoyl CoA into TG. As shown in Table 2, taxifolin added to the culture medium for 24 h decreased the rate of incorporation of [14 C]palmitoyl CoA into TG. Percent inhibition was 35% (n = 3, P < 0.05 versus untreated control). These results compared well with our previous finding on quercetin [4] and correlated well with our results on microsomal TG synthesis.

3.2. Taxifolin decreases microsomal triglyceride synthesis and its subsequent accumulation in the lumen

3.4. DGAT inhibition by taxifolin is non-competitive

We followed our study by examining whether the decreased in apoB secretion was due to limited lipid availability. Lipid availability, particularly TG, is a predominant factor in determining the rate of apoB secretion in HepG2

To characterize DGAT inhibition, the activity of taxifolin on enzymatic activity was assessed. The rate of reaction of DGAT was measured at various concentrations of substrate (i.e., palmitoyl CoA; 2–10 ␮mol/L) in the presence

Table 1 Effects of taxifolin on the cellular distribution of newly synthesized triglyceride Treatment

Cytosol (nmol [3 H]TG/mg cell protein)

Lumen (nmol [3 H]TG/mg cell protein)

Membrane (nmol [3 H]TG/mg cell protein)

OA control OA + taxifolin

4.6 ± 0.7 2.8 ± 0.6*

1.9 ± 0.2 1.4 ± 0.03*

2.4 ± 0.4 1.5 ± 0.3*

HepG2 cells were pre-treated with taxifolin (200 ␮mol/L) for 22 h in a SF media containing BSA/oleic acid (OA) (0.81 mmol/L). Cells were then labeled with 10 ␮Ci/mL [3 H]glycerol ± taxifolin + BSA/OA for an additional 2 h. Cells were then homogenized and subcellular fractions (i.e., cytosol, microsomal membrane, and microsomal lumen) were isolated. TG in each fraction was extracted and separated by TLC. The amount of [3 H]TG was determined by scintillation counting of the TG spot. Data are expressed as nmol of [3 H]glycerol incorporated into TG per mg of cell protein. Values represent the mean ± S.D. of three independent experiments performed in duplicate. *P < 0.05 vs. OA control.

A. Casaschi et al. / Atherosclerosis 176 (2004) 247–253 Table 2 Effects of taxifolin on DGAT activity Treatment

DGAT activity (pmol [14 C]TG/mg cell protein/min)

Control Taxifolin

8170 ± 160 5310 ± 280*

HepG2 cells were pre-treated with taxifolin (200 ␮mol/L) for 24 h in SF media containing BSA/oleic acid (0.81 mmol/L). Cell lysates were prepared and DGAT activity was determined by esterification of diacylglycerol, using [14 C]palmitoyl CoA. The amount of [14 C]TG was determined by TLC and scintillation counting. Data are expressed as pmol of [14 C]palmitoyl CoA incorporated into TG per mg of cell protein per minute. Values represent the mean ± S.D. of three independent experiments performed in duplicate. *P < 0.05 vs. control.

or absence of taxifolin (200 ␮mol/L). Taxifolin was added directly to the DGAT assay reaction mixture containing control HepG2 microsomes and substrates for 10 min. The Lineweaver–Burke plot showed a non-competitive pattern of inhibition. There was a dose-dependent decrease in the Vmax value with taxifolin (77% at 200 ␮mol/L versus untreated control) while the Km value (approximately 7 ␮mol/L) remained essentially unchanged in the absence and presence of taxifolin (Fig. 3). The result suggests that the inhibitory effect on DGAT activity by taxifolin is non-competitive in nature. The experiment was repeated twice with similar results. 3.5. Taxifolin has no effect on DGAT-1 and DGAT-2 mRNA expression In order to further characterize the decrease in DGAT activity, we measured the expression levels of DGAT-1 and

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-2 mRNA in response to taxifolin treatment. Two forms of DGAT (i.e., DGAT-1 and DGAT-2) have been identified [18]. Cells were treated with 100 and 200 nmol/L taxifolin for 24 h in the presence of oleate. Total RNA was extracted from the treated groups and untreated control, and relative RT-PCR was performed to detect the transcript level of DGAT-1 and -2 gene. Fig. 4 is the ethidium bromide stained gel of a typical experiment performed in duplicate (reproduced in two other experiments in duplicate). When normalized to the 18S rRNA internal control, taxifolin was shown to have no significant effect on DGAT-1 (+3% at 100 ␮mol/L and +8% at 200 ␮mol/L versus untreated control) and DGAT-2 (+4% at 100 ␮mol/L and −6% at 200 ␮mol/L versus untreated control) mRNA expression. The result suggests that under our condition, DGAT activity is not regulated at the transcriptional level, but rather post-transcriptional. 3.6. Taxifolin decreases MTP activity MTP is mainly known to aid in the transfer of lipids to the nascent apoB molecule to form the primordial lipoprotein. Evidence is also accumulating to suggest that MTP may be involved in the accumulation of TG in the ER lumen required for “core expansion” of the nascent primordial particle (reviewed in [19]). Both actions are considered major determining factors in the assembly and secretion of apoB-Lp. Since, taxifolin was shown to limit TG accumulation in the lumen, we continued our investigations by examining the effects of taxifolin (200 ␮mol/L) on MTP activity in the presence of oleate. Using a fluorogenic-labeled donor liposomes and phospholipid acceptor liposomes, cells treated with taxifolin reduced MTP activity significantly (Fig. 5). Percent inhibition was 41 ± 5% (n = 3, P < 0.05 versus untreated control). Our finding suggests that taxifolin, in part via MTP activity, inhibited apoB secretion by limiting TG accumulation within the microsomal lumen and/or reducing the transfer of nascent TG to apoB to form the primordial lipoprotein particle.

4. Discussion

Fig. 3. Lineweaver–Burke plot for diacylglycerol acyltransferase (DGAT) inhibition. Taxifolin (200 ␮mol/L) was added directly to the DGAT reaction assay mixture containing control HepG2 cell microsomes and various concentrations of substrate. The y-axis (1/v) is expressed as ␮mol TG/mg of microsomal protein/min. The plot represents a typical experiment repeated twice.

Hypertriglyceridemia plays a crucial role in the development of atherosclerosis and is a common symptom in subjects with insulin resistance and obesity [20,21]. A major contributor to hypertriglyceridemia is the hepatic overproduction of VLDL [22]. While the mechanistic framework for understanding hepatic VLDL overproduction has evolved dramatically in the past decade (reviewed in [1,23]), information on the role of DGAT on VLDL-apoB production is lacking. We recently reported that flavonoids have the ability to inhibit DGAT in the intestinal cell-line, CaCo-2 [4]. While the study was largely descriptive in nature, the mechanism by which flavonoid influenced DGAT activity and the role of

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Fig. 4. Effects of taxifolin on DGAT-1 and DGAT-2 mRNA levels. HepG2 cells were incubated with taxifolin (100 and 200 ␮mol/L) for 24 h in a SF media containing BSA/oleic acid (0.81 mmol/L). Total RNA was extracted from the cells, mRNA levels analyzed by relative RT-PCR, and the gel stained with ethidium bromide. Band intensities were scanned densitometrically and DGAT-1 and -2 mRNA levels were normalized with respect to the 18S rRNA internal control. The figure is a representative stained gel (with an inversion display) showing the signals to DGAT-1, DGAT-2, and 18S rRNA.

DGAT in the regulation of hepatic apoB secretion remained unexplored. In the present study, we attempted to address the mechanism of action more closely. We first confirmed the effects of taxifolin on apoB secretion. Our results showed that taxifolin is able to inhibit apoB secretion under basal and lipid-rich conditions up to 63%. The effect was specific as albumin secretion remained essentially unchanged. The results compared well with our previous findings on apoB secretion in HepG2 cells [3]. Using pulse-chase experiments, an average reduction of 61% in labeled apoB in the medium was apparent with taxifolin. Upon closer analysis, the reduction in apoB secretion was due to enhanced intracellular protein degradation [3]. The availability of lipid, particularly TG, is widely accepted as a major contributing factor in the regulation of apoB-Lp assembly and secretion in HepG2 cells [12]. It plays a central role in apoB targeting, either for intracellular degradation, or for assembly as lipoprotein particles. In HepG2 cells, the microsomal lumenal TG pool is viewed as the regulatory pool responsible for lipoprotein assembly

Fig. 5. Effects of taxifolin on microsomal triglyceride transfer protein (MTP) activity. HepG2 cells were treated with 200 ␮mol/L of taxifolin in a SF media containing BSA/oleic acid (0.8 mmol/L) for 24 h. Cell homogenates were prepared and MTP activity was determined. Data are expressed as a percentage of control (set as 100%). Values represent the mean ± S.D. of three independent experiments performed in duplicate. * P < 0.05 vs. control.

[17]. This pool is derived from a single site of TG synthesis that is associated with the microsomal membrane. Thus, we examined whether taxifolin may act on apoB secretion by limiting the microsomal synthesis of TG and its subsequent influx into the lumen. We added oleate to all our experiments to overcome the low availability of lipids necessary for lipoprotein assembly [17]. Our results showed that taxifolin is able to reduce microsomal TG synthesis by 35%. The reduction was coupled with a similar reduction in the accumulation of TG into the lumen, suggesting that TG availability may be a major determining factor in the regulation of apoB secretion under our condition. The results compared well with our previous findings on whole cell TG synthesis in HepG2 cells [3]. Although, our previous study on taxifolin was crucial in identifying flavonoids as hypotriglyceridemic agents, little insight was provided as to the mechanism for this action. In the present study, we investigated the effect of taxifolin on cellular DGAT activity, a key enzyme in TG synthesis [11]. Interestingly, we found DGAT activity to be significantly reduced (37%) under taxifolin treatment. To delineate the mechanism for the reduction in DGAT activity, competitive binding assays and mRNA expression analyses were performed. In the competitive binding assay, Vmax decreased considerably while Km remained essentially unchanged under taxifolin treatment, suggesting a non-competitive type of inhibition. Using relative RT-PCR, DGAT-1, and DGAT-2 mRNA, expression levels remained essentially unchanged in the presence of taxifolin ruling out transcriptional regulation. The suppression in DGAT activity is therefore, due to a post-transcriptional mechanism of action. Recently, Yu et al. [24] suggested that DGAT was regulated mainly at the translational level. It is known that MTP, in addition to catalyzing the transfer of lipids to nascent apoB molecules, facilitates the accumulation and attainment of TG within the microsomal lumen [10]. A similar flavonoid, naringenin, was recently shown to inhibit apoB secretion by limiting the accumulation of microsomal TG via MTP inhibition [9]. Thus, it is probable that taxifolin, by inhibiting MTP activity, may also be limiting the transfer and accumulation of microsomal TG into the lumen available for lipoprotein assembly. Thus, we followed our investigation by examining whether the reduction

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in TG availability may also be associated with concomitant decrease in MTP activity. Interestingly, taxifolin was also shown to significantly reduced MTP activity by 41%. This suggests that taxifolin may limit MTP-mediated accumulation of newly synthesized TG within the microsomal lumen. In summary, the data in this report indicated that taxifolin inhibited apoB secretion by limiting microsomal TG synthesis and its subsequent accumulation in the microsomal lumen. Lack of lipidation of the lipoprotein particle is known to divert apoB into a degradative pathway and thus, reduce secretion. Whether the reduction in lumenal TG accumulation is predominantly mediated through DGAT and/or MTP activity remains to be addressed. Specific DGAT inhibitors would be required and presently, none are available. The inhibitory effect of flavonoids on DGAT activity is intriguing. However, since flavonoids inhibit a number of lipogenic enzymes, confirmatory studies, using over-expressed or knockout DGAT genes in cultured hepatocytes would be necessary in extrapolating the role of DGAT on apoB secretion. This study suggests a potential role of plant flavonoids in the treatment of hypertriglyceridemia.

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Acknowledgements Supported by the American Heart Association of Hawaii (0350528Z) and the Robert C. Perry Fund of the Hawaii Community Foundation (20020609). B.R. is a recipient of a fellowship from the National Institute of Health (NIH), MARC U* STAR Program (GM07684-23).

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