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Journal of the American College of Nutrition, Vol. 26, No. 1, 66-75 (2007)
Published by the American College of Nutrition

Flaxseed Reduces Plasma Cholesterol Levels in Hypercholesterolemic Mouse Models

Michael A. Pellizzon, PhD, Jeffery T. Billheimer, PhD, LeAnne T. Bloedon, MS, RD, Philippe O. Szapary, MD and Daniel J. Rader, MD

Institute for Translational Medicine and Therapeutics (M.A.P., J.T.B., L.T.B., P.O.S., D.J.R.)
Division of General Internal Medicine, General Clinical Research Center University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania (P.O.S., L.T.B.)

Address correspondence to: Daniel J. Rader, M.D., Center for Experimental Therapeutics, University of Pennsylvania School of Medicine, 421 Curie Blvd., BRB II/III, Philadelphia, PA 19104-6160. E-mail: rader{at}mail.med.upenn.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Objective: We examined the effects of whole ground flaxseed added to a Western diet on plasma and hepatic lipids and hepatic gene expression in male and female human apolipoprotein B-100 transgenic (hApoBtg) mice which have a plasma lipid profile more closely resembling man than wild type mice and in mice lacking the low density lipoprotein receptor (LDLr) and apolipoprotein B mRNA editing enzyme complex 1 (LDLr–/–/apobec–/–).

Methods: The Westernized control diet containing 0.1% cholesterol and 30% kcal as fat was fed for 10 days to hApoBtg mice and for 14 days to LDLr–/–/apobec–/– mice. Animals from each genetic background were then divided into 2 groups based on gender and mean plasma total cholesterol (TC). The hApoBtg and LDLr–/–/apobec–/– mice either continued on the control diet for a total of 31 and 35 days, respectively or were fed 20% w/w whole ground flaxseed (flax) with comparable caloric, macronutrient and fiber content for 21 days. Blood was obtained after a 4 hour fast from all mice prior to feeding both control and flax diets, after 10 days on the flax diet, and after 21 days on the flax at which time all mice were exsanguinated.

Results: The control diet increased TC by >100 mg/dl in the hApoBtg with a greater increase observed in males and by 800 mg/dl in mice lacking the LDLr. After 3 weeks, the flax diet significantly reduced plasma TC by 19% and 22% in hApoBtg and LDLr–/–/apobec–/–, respectively and non-high density lipoprotein cholesterol (non-HDL-C) by 24% in both models (p for all <0.05). Flax significantly reduced hepatic cholesterol in hApoBtg by 32% and 47% in males and females, respectively and LDLr–/–/apobec–/– mice by 66%. Flax had no effect on the expression of the following hepatic genes: LDLr, 3-hydroxy-3-methylglutaryl (HMG) CoA reductase, phospholipid transfer protein, cholesterol 7{alpha} hydroxylase, fatty acid synthase, and acyl CoA oxidase in either mouse model.

Conclusions: Flaxseed reduces plasma and hepatic cholesterol in hApoBtg mice, but had no effect on hepatic lipogenic genes and was equally effective in mice lacking LDLr. The combined data suggest that the lipid lowering effect of flax is not hepatic mediated and may be at the level of cholesterol absorption and/or bile acid reabsorption.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Data from controlled clinical trials [1–3] suggest whole ground flaxseed and flaxseed meal modestly reduce total cholesterol (TC) and low density lipoprotein cholesterol (LDL-C), yet the mechanism is unknown. Hypercholesterolemia is one of the most important established risk factors for atherosclerotic cardiovascular disease (ASCVD), which continues to be the leading cause of morbidity and mortality in the United States [4]. It is estimated that for each 1% reduction in LDL-C, ASCVD risk is reduced by 1% [5]. Lifestyle interventions, such as diet alone, can modestly lower LDL-C and have a substantial impact on ASCVD mortality at a population level [1,6]. It is estimated that 65.3 million Americans are candidates for lifestyle changes to lower cholesterol [4]. Therefore, investigating dietary components that can significantly improve hypercholesterolemia is important.

Flaxseed is the richest known source of alpha-linolenic acid (ALA) and the phytoestrogen, lignans, as well as being a good source of soluble fiber, all of which have been purported to reduce serum cholesterol [7]. Flaxseed and its individual components are increasing in popularity as functional foods and dietary supplements [7]. Recent marketing data reveals flaxseed sales have increased significantly since 2001 and remain second only to fish oil supplement sales [8].

The mechanism of the observed hypolipidemic effect of flaxseed is yet unknown. It is difficult to study factors that affect LDL-C in wild type mice because the majority of plasma cholesterol is in HDL. Due to limitations of established animal models in studying human hypercholesterolemia, we chose to investigate hypolipidemic-related mechanism(s) using human apolipoprotein B transgeneic (hApoBtg) mice, which overexpress human apolipoprotein B-100 and have elevated LDL-C. Additionally, because of the pivotal role the LDLr plays in modulating LDL-C, similar experiments were performed with male mice which lack both LDLr and apolipoprotein B editing enzyme complex 1, which is characterized as an authentic model of human familial hypercholesterolemia [9].


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 ACKNOWLEDGMENTS
 REFERENCES
 
Animals and diets
C57B1/6 hApoBtg mice (obtained from Dr. S. Young) and C57B1/6 LDLr–/–/apobec–/– mice were bred in house and have been described elsewhere [10,11]. Adult hApoBtg male (n = 14) and female mice (n = 25) (aged >2 months) and male LDLr–/–/apobec–/– mice (n = 11) were housed in polycarbonate cages (up to 4 animals per cage) in a room with a 12-hour light/dark cycle (lights on at 600 h), temperature of 22 ± 1°C, and humidity of 30–50%.

For all experiments, diets were prepared by Research Diets, Inc. (New Brunswick, NJ) and contained AIN-93M [12] vitamin and mineral mixes (Table 1). The control diet was created to represent a Western diet that contained the typical US dietary fatty acid composition (Table 2) as well as 0.1% cholesterol and 30% kcal as fat. The experimental diet was the flaxseed diet (flax diet), consisting of whole ground flaxseed (20% w/w) where adjustments were made to the casein, fat, and cellulose contents in order to produce diets with similar caloric, macronutrient and fiber contents (Table 1). Whole ground yellow Omega flaxseed, which was grown, harvested, and processed in North Dakota was provided free by Dr. Jack Carter (North Dakota Oil Seed Council and the Flax Section of North America, Bismark, ND) and contained (w/w): 42.4% total lipids (53.3% ALA, 17.2% linoleic acid, 18.6% oleic acid, 3.3% stearic acid, 4.7% palmitic acid), 25.3% total carbohydrate (of total carbohydrate, 6.7% soluble fiber, 17.9% insoluble fiber), 19.7% protein, and 1.6% lignans.


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  		Table 1. Nutrient Composition of the Control and Flax Diets

 

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  		Table 2. Fatty Acid Profile for the Control and Flax Diets1

 
Procedure
All animal experiments were performed using the same feeding protocol. After an initial fasting (4 hr) blood draw from the retro-orbital sinus vein, hApoBtg mice were fed the control diet for an introductory period of 10 days to acclimate animals on the purified diet and to elevate their plasma TC levels. Male LDLr–/–/apobec–/– mice in a subsequent study were on the control diet for 14 days. The period of time of the lead in phase was lengthened for these animals as plasma cholesterol levels of LDLr–/–/apobec–/– are known to respond robustly to a Western diet [9]. On the last day of the introductory control diet run-in, a fasting blood draw was obtained from all animals to determine plasma TC levels. Based on gender and plasma TC at the end of the control diet run-in, hApoBtg mice and LDLr–/–/apobec–/– mice were assigned to the flax or control diet (male hApoBtg: 7 mice/treatment; female hApoB, n = 13 on the flax diet and n = 12 on the control diet; LDLr–/–/apobec–/–, n = 6 on flax diet and n = 5 on control diet) for 21 days. Blood was obtained from all animals on days 0, 10 and 21. Animals were exsanguinated and livers harvested on day 21. Plasma was obtained from heparinized blood and frozen at –20°C for future analysis. The left lobe and caudate lobes of mouse livers were flash frozen in liquid nitrogen and stored at –80°C prior to the measurement of tissue lipids and RNA isolation. The study protocol was approved by the Institutional Animal Care and Use Committee at The University of Pennsylvania.

Plasma Lipid and Lipoprotein Analysis
Plasma total, free, and high density lipoprotein (HDL) cholesterol; phospholipid, non-esterified fatty acids, and triglyceride (TG) levels were measured enzymatically on a Cobas Fara II (Roche Diagnostic Systems Inc.) using Sigma reagents (Sigma Chemical Co., St. Louis, MO). Plasma phospholipid fatty acid composition was performed by Nutrasource Diagnostics, Inc. (Guelph, Ontario, Canada).

Fast Protein Liquid Chromatography (FPLC) Analysis
Plasma lipoproteins were characterized using gel filtration on FPLC. Pooled plasma samples (120 µL) were subjected to FPLC gel filtration (Pharmacia LKB Biotechnology, Uppsala, Sweden) on two Superose 6 columns at a flow rate of 0.5 ml/min (1 run/sample). Cholesterol was determined using an enzymatic assay (Wako Pure Chemical Industries Ltd., Osaka, Japan).

Hepatic Cholesterol and Triglyceride Analysis
Liver was homogenized in a saline solution (~250 mg liver/ml saline). Cholesterol and triglyceride were measured as described [13] after solubilization of lipid by deoxycholate using cholesterol reagent or triglyceride reagent and lipid lintrol standards.

RNA Isolation and Quantitative Real-Time Polymerase Chain Reaction (PCR) Analysis
Total RNA were isolated using the Qiagen RNeasy Mini kit and any contaminating DNA was removed with the Qiagen RNase-free DNase set (Qiagen, Valencia, CA). Quality of the isolated RNA was controlled for by the 260:280 nm ratio.

For quantitative real-time PCR analysis, cDNA was synthesized from 1 µg of total RNA by reverse transcription using the High-Capacity cDNA Archive Kit (Applied Biosystems, Foster City, CA). Primers and probes for all genes were designed using Primer Express 2.0 software (Applied Biosystems, Foster City, CA) and purchased from Qiagen (Valencia, CA). For HMG CoA reductase, Sybr® Green PCR Master Mix (Applied Biosystems, Foster City, CA) was used in place of the TaqMan probe. Probes are double-labeled with a 6-FAM reporter dye at the 5' end and a Tamra-Q quencher dye at the 3' end. The reactions were performed using MicroAmp Optical 96-well plates with optical adhesive covers (Applied Biosystems, Foster City, CA). The primers and probe for each gene were added in appropriate concentrations to a 2X TaqMan buffer containing AmpliTaq Gold®, 10X PCR Buffer II, and MgCl2 buffer. A total of 1 µl of cDNA of each unknown sample was added to each well. In order to determine the mRNA expression of unknown samples, a relative quantification of the mRNA expression was performed using data from a highly expressing hepatic cDNA sample, which was serially diluted 10 fold. The expression data of target genes was normalized with 18S rRNA (primers and TaqMan probe, Applied Biosystems, Foster City, CA). Relative mRNA expression of the experimental diets was reported as a fold change from mRNA levels of the control diet. All quantitative real-time PCR analysis was performed using an ABI 7300 (Applied Biosystems, Foster City, CA).

Statistical Analysis
All statistical analyses were performed using JMP version 5.0.1a (SAS Institute, Inc., Cary, NC). Effect of diets at each time point for plasma lipids were assessed using unpaired t-tests comparing % change at day 10 and day 21 between groups. These analyses were confirmed using a repeated measures analysis of variance (ANOVA) techniques in which terms for treatment (flax vs. control) and time (days 0, 10, 21) were used. Because we were interested in specific gender lipoprotein responses, we also added a gender term in these ANOVA models. All liver data were assessed using unpaired t-tests. Because significant interactions were noted for plasma lipid parameters, results were displayed separately for both genders. Significance was set at p < 0.05.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
ACKNOWLEDGMENTS
 REFERENCES
 
Plasma Lipids
Since we found significant differences between male and female hApoBtg mice with regards to HDL-C, results are reported separately. In particular, we found a significant gender by treatment interaction in terms of HDL-C (p = 0.02) at 21 days.

Male hApoBtg Mice.
The 10 day run-in control diet increased plasma TC, non-HDL, and HDL levels by 150%, 230%, and 24%, respectively with no effect on TG concentrations (Fig. 1). From day 0 to 21, there was no further change in any lipid parameter in the control group. Flaxseed reduced TC, non-HDL-C and HDL-C by day 10 and at 21 days, produced net reductions of 19%, 24%, and 7%, respectively, with no affect on plasma TG concentrations relative to the control group. The reductions in plasma TC were reflected mainly by reductions in the non-HDL-C fraction (net decrease of 34%, p < 0.05). Repeated measures ANOVA confirmed the results presented above. Fractionation of the plasma by FPLC corroborates that the main change in plasma cholesterol is in the LDL-C fraction (non-HDL-C by enzymatic method) (Fig. 2), Panel A.


Figure 1
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  		Fig. 1. Plasma total cholesterol (panel A), non-HDL-C (panel B), HDL-C (panel C), and triglyceride (panel D) levels throughout the study for male hApoBtg mice fed either control (n = 7) or flax diets (n = 7).

 

Figure 2
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  		Fig. 2. FPLC fraction cholesterol levels on day 21 in male (panel A) and female (panel B) from pooled plasma samples in hApoBtg mice fed either control or flax diets.

 
Female hApoBtg Mice.
During the 10 day run-in, the control diet increased plasma TC, non-HDL and HDL-C by 81%, 108%, and 28%, respectively, and reduced TG by 38% (Fig. 3). After 21 days, the flax diet reduced plasma TC by 18% and non-HDL-C by 27% relative to the control diet without affecting HDL-C or TG. Repeated measures ANOVA confirmed the results presented above. Pooled plasma cholesterol levels fractionated by FPLC on day 21 confirmed that reductions were in the LDL fraction (Fig. 2, Panel B).


Figure 3
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  		Fig. 3. Plasma total cholesterol (panel A), non-HDL-C (panel B), HDL-C (panel C), and triglyceride (panel D) levels throughout the study for female hApoBtg mice fed either control (n = 13) or flax (n = 12) diets.

 
LDLr–/–/apobec–/– Mice
During the 14 day run-in, the control diet increased plasma TC, non-HDL-C, HDL-C, and TG by 190%, 230%, 103%, and 38%, respectively (Fig. 4). After the run in period, the control diet further increased plasma TC, non-HDL-C, HDL-C and TG by 22%, 22%, 23%, and 45%, respectively over the next 21 days. The flax diet reduced TC, non-HDL-C and HDL-C by day 10 and at 21 days, produced net reductions of 22%, 24%, and 25%, respectively. Although the flax diet had produced significantly lower plasma TG levels at day 21, the levels were not significantly reduced over time (Fig. 4) compared to the control diet. While the absolute changes in plasma TC and non-HDL-C from days 0 to 21 were greater than those of hApoBtg mice due to the lack of LDL receptors, the percent reductions were similar. Pooled plasma FPLC cholesterol data indicated that cholesterol levels in both VLDL and LDL fractions were reduced by the flax diet (Fig. 5).


Figure 4
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  		Fig. 4. Plasma total cholesterol (panel A), non-HDL-C (panel B), HDL-C (panel C), and triglyceride (panel D) levels throughout the study for male LDLr–/–/apobec–/– mice fed either control (n = 6) or flax (n = 5) diets. *Different from control diet fed mice, p < 0.05.

 

Figure 5
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  		Fig. 5. FPLC fraction cholesterol levels on day 21 from pooled plasma samples in male LDLr–/–/apobec–/– fed either control or flax diets.

 
Hepatic Lipids
In hApoBtg mice, flax significantly reduced the hepatic cholesterol content by 32% in males and 47% in females (Fig. 6), but had no significant affect on hepatic TG levels. In LDLr–/–/apobec–/– mice, flax significantly reduced both hepatic cholesterol and TG levels by 66%.


Figure 6
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  		Fig. 6. Hepatic lipid levels on day 21 in male (panel A) and female (panel B) hApoBtg mice and of male LDLr–/–/apobec–/– mice (Panel C) fed either control or flax diets. *Different from control diet fed mice, p < 0.05. Male hApoBtg: 7 mice/treatment; female hApoB: Flax, n = 13 and Control, n = 12; LDLr–/–/apobec–/–: Flax, n = 6 and Control, n = 5.

 
Hepatic mRNA Expression
Relative to the control diet, the flax diet did not influence the relative expression of mRNA of the LDL receptor (LDLr), HMG-CoA reductase, phospholipid transfer protein (PLTP), cholesterol 7{alpha} hydroxylase (cyp7a), fatty acid synthase (FAS), and acyl CoA oxidase (ACO) were not influenced by flax in hApoBtg mice (flax and control data, respectively: LDLr, 1.0 ± 0.1 and 1.0 ± 0.5, p = 1.0; HMG CoA reductase, 1.1 ± 0.1 and 1.0 ± 0.1, p = 0.29; Cyp7{alpha}, 1.0 ± 0.5 and 1.1 ± 0.4, p = 0.67; FAS, 1.0 ± 0.4 and 0.9 ± 0.2, p = 0.71; ACO, 1.0 ± 0.3 and 1.1 ± 0.3, p = 0.76; PLTP, 1.0 ± 0.3 and 0.9 ± 0.1, p = 0.28) and results were also similar in LDLr–/–/apobec–/– mice (data not shown).

Plasma Phospholipid Fatty Acid Profile
Pooled plasma from both groups at 21 days for male hApoBtg mice was obtained for the measurement of phospholipid fatty acid profile. The percentages of plasma phospholipids as total SFA, monounsaturated fatty acid (MUFA), and PUFA ({omega}-3 and {omega}-6) were similar between the two groups (control and flax diet, respectively: SFA = 44.0% and 43.2%; MUFA = 15.9% and 14.0%; total PUFA = 40.0% and 42.8%). Flaxseed increased ALA, 20:5, {omega}-3 eicosapentaenoic acid (EPA), and total {omega}-3 PUFA but decreased 22:6, {omega}-3 docosahexaenoic acid (DHA) relative to the control diet (control and flax diet, respectively: ALA = 0.1% and 1.1%, EPA = 1.7% and 2.4%, total {omega}-3 PUFA = 8.8% and 10.0%, and DHA = 6.6% and 5.0%). Flax had no effect on the ratio of {omega}-3/{omega}-6 (control and flax diet were 3.5 and 3.3, respectively).


   DISCUSSION
 
Similar to what others have reported for a high fat diet [14], providing hApoBtg male and female mice a Western diet containing a fatty acid profile similar to US intake and 0.1% cholesterol significantly raises TC, non-HDL and HDL-C levels. The percent increase in TC, non-HDL and HDL-C tended to be greater in males than females which may be due to known gender differences in cholesterol absorption and hepatic cholesterol metabolism [15]. We convincingly demonstrate that 20% w/w ground flaxseed significantly reduced plasma TC and non-HDL cholesterol in hApoBtg male and female mice while following the westernized diet for 21 days.

Male mice had a greater reduction in all parameters as compared to females. A possible explanation may be due to the presence of endogenous sex hormones. In a previous study, ovariectomized hamsters have elevated TC and HDL-C levels relative to sham operated control females [16]. The addition of 7.5%–22.5% flaxseed to diets or estrogen injections reduced plasma TC and HDL-C levels of ovariectomized female hamsters back to those of sham operated animals. Because flaxseed contains a high concentration of lignans, which are phytoestrogens with weak estrogenic activity, the addition of flaxseed may provide more hypocholesterolemic activity to males relative to fertile females in whom endogenous estrogens are already at high levels. Alternatively, there may be gender difference in cholesterol absorption that could contribute to differences reported.

Flaxseed significantly reduced plasma HDL-C in male, but not female mice when measured enzymatically. A negative correlation between ALA and HDL-C has been demonstrated in 2 recent randomized controlled trials. Wilkinson et al [17] randomized 57 men to a diet either enriched with ALA from flaxseed oil (19 g ALA/d), linoleic acid (LA) from sunflower oil, or sunflower oil with fish oil for 12 weeks. Plasma HDL-C was reduced by 10.5% in the flax oil group compared to a reduction of 5.6%, and an increase of 3% in the sunflower and sunflower + fish oil groups, respectively (p = 0.009). Another recent feeding study compared an average American diet, a high ALA diet, and a high LA diet [18] supplying 36% energy as total fat and found a significant reduction in HDL-C of ~6% in 23 subjects (20 of whom were male) following the high ALA diet compared to the average American diet. The mechanism of this apparent ALA induced reduction in HCL-C in men is not known and neither is its clinical significance.

In contrast to the enzymatic data, analysis of pooled plasma cholesterol levels fractionated by FPLC revealed no gender difference in HDL-C. The reason for this is not immediately apparent, however, it could reflect an inability to precipitate all of the ApoB containing lipoproteins at high total plasma cholesterol. This would yield a higher than expected HDL-C value in the enzymatic analysis but not by FPLC separation. Nevertheless both enzymatic and FPLC analyses indicated that LDL was the major lipoprotein fraction reduced by flaxseed. The LDL fractional peak was not shifted to the right or left for either males or females. This suggests that the particle size was not greatly affected.

In addition to reducing plasma cholesterol, flaxseed significantly reduced hepatic cholesterol in both male and female hApoBtg mice. Although significant changes were observed in both plasma and hepatic cholesterol levels, the mRNA expression levels of hepatic genes involved in cholesterol metabolism, including those involved in cholesterol synthesis (HMG CoA reductase), uptake (LDLr), bile acid synthesis (cyp7{alpha}), and VLDL secretion (PLTP) [19] were not influenced. The lack of a significant change in the expression of HMG CoA reductase may not have be entirely unexpected. It is known that an increased cholesterol intake reduces mRNA expression levels for HMG-CoA reductase. The level of cholesterol intake in the current study far exceeds hepatic cholesterol synthesis per day for a mouse according to Dietschy et al [20] (cholesterol synthesis = ~50 mg/day per kg, current study cholesterol intake = ~125 mg/day per kg). In addition, the level of hepatic cholesterol was still elevated above levels of wild type mice fed rodent chow, which typically contains ~0.02% cholesterol (w/w) (Hepatic cholesterol levels: chow fed wild type mice, 2.74 ± 0.39 mg/g). Therefore, even in mice receiving the flax diet the concentration of hepatic cholesterol is still sufficient to downregulate HMG CoA reductase.

Although no significant changes were seen in the expression of these hepatic genes, this does not rule out post-translational effects. Because approximately 88% of LDL-C turnover per day is attributable to the LDLr in the mouse [21], and only 12% is receptor independent, we determined whether an intact LDL receptor was required for the cholesterol lowering activity of flaxseed. We chose a mouse model that had a targeted deletion of the LDLr and expresses high levels of apoB-100 (LDLr–/–/apobec–/–). These mice had a targeted deletion of the apobec-1 gene, which would inhibit the truncation of apoB-100 to form apoB-48, resulting in plasma LDL associated with apoB-100 only (9). While Apo B48 can be cleared by the ApoE receptor, apoB-100 requires the LDLr for clearance. Flaxseed reduced the plasma non-HDL-C from day 0 to day 21 in male LDLr–/–/apobec–/– mice by a percentage comparable as seen in the hApoBtg male mice, indicating that the reduction in apoB containing lipoproteins was independent of the LDLr.

Flaxseed also contains ~7% soluble fiber. Soluble fiber from some grains have been shown to interact with cholesterol in the intestinal lumen to decrease its absorption while others appear to inhibit the reabsoption of bile acids [22–24], either of which would result in a lowering of plasma cholesterol in mice fed a diet higher in cholesterol. Since in these studies flax did not affect hepatic lipogenic enzymes, lipid absorption is the most likely mechanism of its hypocholesterolemic action and are a subject of future studies.

In addition to cholesterol regulating genes, we also measured the mRNA abundance of hepatic genes key to the regulation of fatty acid synthesis and oxidation. This was of interest because flax significantly increased ALA (18:3, {omega}-3 PUFA) and EPA (20:5, {omega}-3 PUFA) plasma phospholipids in hApoBtg mice. An increased intake of omega 3 fatty acids has been shown to reduce the hepatic mRNA expression of fatty acid synthesis enzymes (i.e. FAS) and increase that of fatty acid oxidation enzymes (i.e. ACO) [25,26]. However, flax supplementation had no significant effect on the mRNA expression of FAS and ACO, which is consistent with a lack of any significant effect on plasma and hepatic TG levels in hApoBtg mice. The data by Ide et al [27] showed that the hepatic mRNA expression of FAS was reduced and the expression of ACO was increased to a greater extent by fish oil (high in EPA and DHA) relative to perilla oil (high in ALA) [26]. Because the flax diet increased total {omega}-3 PUFAs only slightly (12%) and actually decreased EPA + DHA slightly (11%) in plasma phospholipid, this suggested that these modest changes induced by the flax to the phospholipid fatty acid profile may not be adequate to have any significant influence on the mRNA expression of genes involved in fatty acid synthesis or oxidation.

In summary, while flaxseed has been shown to reduce plasma cholesterol levels in rats [28] and hamsters [16], the current study is the first to show that the addition of 20% w/w whole ground flaxseed to a Western diet with the typical U.S. fatty acid profile and 0.1% cholesterol significantly reduces plasma non-HDL-C levels in hApoBtg mice, a mouse model with a lipoprotein profile similar to that of humans. Flaxseed also significantly reduced plasma cholesterol in the LDLr–/–/apobec–/– mouse, indicating that the hypocholesterolemic influence of flaxseed was independent of the LDLr and therefore, ruled out a major cholesterol clearance pathway. Alterations in plasma cholesterol levels by flaxseed were not related to any significant effect on the expression of hepatic genes important to cholesterol metabolism. Future studies are necessary to determine the molecular mechanisms of flax-induced cholesterol reduction and the hypocholesterolemic potential of individual components in flaxseed, including soluble fiber, ALA, and lignans.


   ACKNOWLEDGMENTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 ACKNOWLEDGMENTS
REFERENCES
 
We would like to thank Anna Lillethun, Linda Morrell, Debra Cromley, Aisha Faruqi, and Anthony Secreto for excellent technical assistance. DJR is a recipient of a Burroughs Wellcome Fund Clinical Scientist Award in Translational Research. MAP was supported by an Institutional Training Grant (HL07443). POS was supported in part by the following grants from the NIH: R21AT01292 (POS), K-23 AT-00058 (POS). We also would like to thank Dr. Jack Carter for his donation of ground flaxseed.

Received October 21, 2005. Accepted February 14, 2006.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 ACKNOWLEDGMENTS
 REFERENCES
 

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