high-cholesterol diets

Nutrition Research 26 (2006) 573 – 578 www.elsevier.com/locate/nutres

Ethanolamine improves hypercholesterolemia in rats fed high-fat/high-cholesterol diets Hisae Kume4, Keiko Tsukahara, Keiko Okazaki, Hajime Sasaki Nutritional Research Department, Food Science Institute, Meiji Dairies Corporation, Odawara 250-0862, Japan Received 25 June 2006; accepted 25 July 2006

Abstract It is known that ethanolamine (Etn) decreases serum cholesterol level in rats fed a cholesterol-free diet and that bezafibrate (Bf) is effective in hypercholesterolemia. In this study, we examined the effects of Etn on serum and liver lipid in rats fed a high-fat/high-cholesterol diet and compared them to the effects of Bf. In addition, we measured the mRNA expression of apoA-I, apoB, and apoE in the liver and small intestine. Hypercholesterolemic rats fed a high-fat/high-cholesterol diet with water were included in the control group, and groups administered 0.25, 0.5, and 1 mg/mL Etn containing water were the experimental groups; the rats were fed for 8 days. The positive control group diet contained 0.1% Bf. Ethanolamine decreased very low-density lipoprotein (VLDL) cholesterol and low-density lipoprotein (LDL) cholesterol in a dose-dependent manner, whereas the serum high-density lipoprotein (HDL) cholesterol and the hepatic cholesterol levels were unaffected. The decrease in cholesterol in serum and liver after Bf intake was even greater. Northern blot analysis showed that mRNA expression of apoB was suppressed by more than 50% in liver of rats in the Etn and Bf groups; however, mRNA expression of apoE in liver and of apoA-I and apoB in intestine were unaffected in the rats that were fed with Etn only. The results indicate that Etn improves hypercholesterolemia and might decrease serum cholesterol via the suppression of apoB mRNA in the liver. D 2006 Elsevier Inc. All rights reserved. Keywords:

Ethanolamine; Bezafibrate; Cholesterol; apoB; Hypercholesterolemia

1. Introduction Ethanolamine (Etn) and phosphoethanolamine are present in breast and cow milk [1], but their nutritive and physiological functions have yet to be fully elucidated. In a series of previous experiments, we found that Etn is a nutritional factor used to synthesize phosphatidylethanolamine (PE) in hepatocytes and to regulate hepatocyte proliferation in vivo and in vitro [2-5]. Ethanolamine is used to synthesize PE and phosphatidylcholine via the methylation of PE by phosphatidylethanolamine N-methyletransferase (PEMT) in hepatocytes [6-8], and the PEMT 4 Corresponding author. Tel.: +81 465 37 3661; fax: +81 465 37 3624. E-mail address: [email protected] (H. Kume). 0271-5317/$ – see front matter D 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.nutres.2006.09.017

pathway is related to the regulation of serum lipid and fatty acid composition in vivo and in vitro [9,10]. These findings indicate that Etn may also regulate serum lipid levels in hapatocytes. Imaizumi et al [11,12] found that PE derived from soybeans decreased the cholesterol level in serum and liver in rats fed a low-fat/cholesterol-free diet. They also reported that Etn derived from PE decreased the serum cholesterol level but not liver cholesterol. The mechanism by which Etn increases liver microsomal PE and changes the composition of fatty acids in phospholipids has been identified [10]. We identified milk-derived phospholipids that contained mainly PE, phosphatidylcholine, and sphingomyelin decreased cholesterol levels in serum and liver in rats fed a high-fat/high-cholesterol diet (HF/HC diet) [13].

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However, the effect of Etn in rats fed an HF/HC diet has not yet been identified. Bezafibrate (Bf) is effective for treating hypertriglyceridemia and hypercholesterolemia in the human and rat [14,15]. It is also an inhibitor of the PEMT pathway, and it decreases very low-density lipoprotein (VLDL) secretion in hepatocytes cultured in the presence of Etn; apoB is also decreased in secreted VLDL from the liver [16,17]. Furthermore, it has been shown in overexpression of apoE, as well as apoB in knockouts, that the amount of apolipoproteins (apoA-I, apoB, and apoE) in liver influences the plasma cholesterol level, in VLDL, low-density lipoprotein (LDL), and high-density lipoprotein (HDL) [18,19]. There appears to be a relationship between the expression of apolipoproteins in the liver and the PEMT pathway and serum cholesterol levels. In this study, we examine the effect of Etn in rats fed an HF/HC diet and compare them with the effect of Bf on rats fed the same diet. We also measured the mRNA expression of apoA-I, apoB, and apoE in the liver and small intestine and found that Etn and Bf decreased serum cholesterol levels, and that there was a relationship between mRNA expression and serum cholesterol levels.

Table 1 Composition of the HF/HC diet Ingredient Casein l-Cystin Mineral mixturea Vitamin mixtureb Choline bitartrate Cellulose powder a-Cornstarch Cornstarch Sucrose Soy oil Lard Cholesterol Sodium cholete

(%) 14.00 0.18 3.50 1.00 0.25 5.00 15.50 35.37 10.00 4.00 10.00 1.00 0.20

a

AIN-93M. Mineral mixture: CaCO 3 35.7, KH 2 PO 4 25.0, K3C6H5O7d H2O 2.8, NaCl 7.4, K2SO4 4.66, MgO 2.4, FeC6H5O7d XH2O 0.606, ZnCO3 0.165, MnCO3 0.063, CuCO3d Cu(OH)2d H2O 0.0324, KlO3 0.001, Na 2 SeO 4 0.001025, (NH 4 ) 6 Mo 7 O 2 4 d 4H 2 O 0.000795, Na2SiO3d 9H2O 0.145, CrK(SO4)2d 12H2O 0.0275, LiCl 0.00174, H3BO3 0.00815, NaF 0.00635, NiCO3d 2Ni(OH)2d 4H2O 0.00306, NH4VO3 0.00066 g/100 g (sucrose + all minerals). b AIN-93 (oriental yeast). Vitamin mixture: nicotinic acid 300 mg, calcium pantothenate 160 mg, pyridoxine hydrochloride 70 mg, thiamine hydrochloride 60 mg, riboflavin 60 mg, folic acid 20 mg, d-biotin 2.0 mg, vitamin B12 250 mg, vitamin E 1,500 mg, vitamin A 40,000 IU, vitamin D3 10,000 IU, vitamin K1 7.5 mg/100 g (sucrose + all vitamins).

2. Methods and materials 2.1. Animals and diets Five-week-old male Sprague-Dawley rats were obtained from Charles River, Hamamatsu, Japan, and maintained on a commercial nonpurified diet (CRF-1, Oriental Yeast Co, Ltd, Tokyo, Japan) for a week before initiation of the experiments with the HF/HC diet. Rats were individually housed in an air-conditioned room at 21.0 F 2.08C and a humidity level of 55.0% F 15.0% with lights on from 7:00 am to 7:00 pm during the experiment. The animal protocol and use of rats was approved by the Food Science Institute of Meiji Dairies Corporation, which follows the Guide for the Care and Use of Laboratory Animals (NRC1996). The HF/HC diet ingredient composition was as follows (wt%): casein, 14; corn starch, 50.87; sucrose, 10; cellulose powder, 5; lard, 10; soybean oil, 4; cholesterol, 1; mineral mixture, 3.5; vitamin mixture, 1; sodium cholete, 0.2; choline bitartate, 0.25; and l-cystin, 0.18 (Table 1). Rats were fed this diet for 7 days, and then 5 groups of 5 rats each were placed on treatment based on serum cholesterol levels and body weight. The first group (control group) was fed a diet with just water for 8 days. Next, 3 experimental groups were fed diets with water containing 0.25, 0.5, and 1 mg/mL Etn (ethanolamine hydrochloride; Sigma, St. Louis, Mo). The last group (positive control group) was fed a diet that included 0.1% Bf (Sigma). Blood was withdrawn from the tail vein 6 days after starting the experiment to determine interim serum cholesterol levels. On the last day, rats were anesthetized by ether, blood was withdrawn from the abdominal main artery, and the liver and small intestine were removed for analysis of liver lipids and Northern blot analysis. Blood was

centrifuged at 10,000  g for 10 minutes, and the supernatant was used for analysis of serum lipids. 2.2. Measurement of lipid composition in serum and liver Total cholesterol, triacylglycerols, and phospholipids in the serum were measured with an enzyme kit (Wako Pure Chemicals, Osaka, Japan), as was LDL cholesterol (Daiichi Pure Chemicals, Tokyo, Japan). HDL cholesterol was measured with a selective blocking kit (Daiichi Pure Chemicals). Total lipids in the liver were extracted with chloroform/ methanol (2:1, V/V) according to the Folch method [20]. The amount of total lipids in the liver was measured after drying the solvents under a stream of N2 gas. The lipid composition was subsequently analyzed using an Iatroscan (Misubishi Kagaku Iatron, Tokyo, Japan), and cholesterolacetate was used as an internal standard. Here, chloroform/methanol/H2O (50:20:25, V/V/V) was used as the first-dimension solvent, and hexane/diethylether/formic acid (65:5:0.15, V/V/V) was used as the second-dimension solvent [21]. 2.3. Northern blot analysis of apoA-I, apoB, and apoE in liver and small intestine Total RNA was extracted from the liver and small intestine using Isogen (Nipon gene, Tokyo, Japan), and mRNA was then extracted using Oligo-dt3 (Takara Shuzou, Tokyo, Japan). Subsequently, 1 lg of mRNA for the detection of apoB or 5 lg tRNA for the detection of apoA-I, apoE, and GAPDH was electrophoresed on an agarose gel and transferred to a membrane (Hybond-N+, Amersham Biosciences,

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2.5. Statistical analysis Results are presented as mean F SEM, n = 6. Statistical analyses were performed using StatView 5.0J (SAS Institute, Cary, NC). Natural logarithmic values were transformed for nonparametric variables. Comparison of multiple groups was carried out by 1-way analysis of variance; when a significant overall effect was detected, differences among individual means were assessed with Fisher multiple comparison test. Results are presented as mean F SEM, n = 6. Differences were considered significant at P b .05. Fig. 1. Changes in serum cholesterol in rats given an HF/HC diet during feeding with 1 mg/mL Etn or 1 mg/g Bf. Values represent mean F SEM, n = 6. Values with different superscripts are significantly different with P b .05 by Fisher-protected least significant difference (PLSD).

3. Results 3.1. The effects of Etn and Bf on lipid composition in serum and liver

Piscataway, NJ). Digoxigenin-labeled probes of apoA-I, apoB, apoE, and GAPDH were prepared using the following primers: apoA-I, 5V-AAG GAC AGC GGC-3V, 5V-GTG AGG CGC CCG-3V; apoB—5V-TCT CGA CTT CCA-3V, 5V-CTG GAG TTG AAG; apoE—5V-CGG AGG CTA AGG-3V, 5V-TAC GCC CTG CCG-3V. Next, the probes were hybridized, reacted with an antidigoxigenin-AP-fragment (Roche, Tokyo, Japan), and detected using CDP-Star (Amersham Biosciences). The membranes were subsequently exposed, developed, and scanned, and signal levels were measured using a National Institutes of Health image.

We examined the effects of Etn in serum and liver lipids in rats fed the HF/HC diet and compared them to those given Bf. As shown in Fig. 1, Table 2, and Table 3, Etn did not influence body weight, food intake, water intake, or liver weight, but liver weight was higher in rats fed the Bfcontaining diet. Ethanolamine decreased cholesterol levels (Fig. 1), VLDL cholesterol, and LDL cholesterol in serum in a dose-dependent manner and improved hypercholesterolemia in rats fed the HF/HC diet (Table 2). However, HDL cholesterol and triacylglycerol in the serum and cholesterol and phospholipids in the liver were not affected. Triacylglycerol levels in the liver tended to be lower in rats given Etn than in those given Bf. Bezafibrate decreased cholesterol and triacylglycerol levels in the serum and cholesterol ester and freecholesterol levels in the liver; however, bezafibrate increased HDL cholesterol levels in the serum and PE and phosphatidylcholine levels in the liver (Tables 2 and 3). Bezafibrate also improved hypercholesterolemia in rats fed the HF/HC diet. The decrease in cholesterol levels in serum after Bf intake was higher than after Etn intake when rats given Etn and rats given Bf were almost the same weight.

2.4. Amino acid analysis Rats were euthanized, and the livers were immediately excised, cut into small pieces with scissors on ice, and homogenized in 150 mmol NaCl, 10 mmol Tris-HCl (pH 7.4), 1.0 mmol EDTA, and 10 mmol NaF. Blood and homogenate were centrifuged at 10,000 rpm for 15 minutes, and supernatants were deproteinized by adding 2 volumes of 5% trichloroacetic acid. Serum was also deproteinized following the same methods. Samples were centrifuged to remove the pellet and filtered through a 0.45-lm filter. Amino acid analysis was performed using an L-8800 Hitachi High Speed Amino Acid Analyzer to measure the Etn and phosphoethanolamine concentrations.

Table 2 Effects of Etn and Bf on body weight, food intake, and serum lipids in rats fed an HF/HC diet for 7 days followed by an experimental diet for an additional 8 days Control Final body weight (g) Food intake (g/day per rat) Water intake (mL/day per rat) Serum (mg/100ml) Total cholesterol ‹ VLDL cholesterol (‹ (› + fi)) LDL cholesterol › HDL cholesterol fi Free cholesterol Triglyceride

Etn (mg/mL)

326.2 F 10.9 20.8 22.8 316.5 203.8 92.3 20.3 53.2 80.0

F F F F F F

27.7a 18.8a 10.5a 2.1a 5.9 24.0

0.25 336.9 F 12.0 21.5 28.2 284.2 183.7 80.8 19.7 45.0 61.5

F F F F F F

26.8ab 18.8ab 8.5ab 1.1a 4.7 4.6

0.5 327.1 F 9.5 20.8 24.1 269.0 174.5 75.3 19.2 46.7 79.0

F F F F F F

20.7ab 17.0ab 5.6ab 2.4a 4.5 24.9

Bf (mg/g) 1.0 313.8 F 11.2 20.1 23.6 223.2 135.7 66.7 20.8 37.0 60.8

F F F F F F

9.3b 6.8b 4.5b 2.0a 1.9 13.8

Values represent mean F SEM, n = 6. Values with different superscripts are significantly different with P b .05 by Fisher-PLSD.

1.0 318.6 F 4.4 18.9 22.8 107.7 52.8 18.8 36.0 37.3 39.2

F F F F F F

9.6c 6.4c 1.6c 2.8b 3.0 5.7

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Table 3 Effects of Etn and Bf on liver weight and liver lipids in rats fed an HF/HC diet Control Liver weight (g) Liver (mg/g liver weight) Total lipid Cholesterol ester Cholesterol Triglyceride Phosphatidylethanolamine Phosphatidylcholine

Etn

21.4 F 1.3

191.0 82.2 5.9 54.7 8.3

F F F F F

Bf

1.0 mg/mL 20.0 F 1.2

17.7a 11.5a 0.2a 11.5 0.4a

188.4 81.5 5.9 43.7 8.2

8.2 F 0.3a

F F F F F

1.0 mg/g 25.3 F 0.8

22.9a 7.3a 0.3a 8.7 0.2a

155.2 48.8 5.2 50.6 10.2

8.2 F 0.07a

F F F F F

17.5b 7.8b 0.2b 17.0 0.3b

11.8 F 0.8b

Values represent mean F SEM, n = 6. Values with different superscripts are significantly different with P b .05 by Fisher-PLSD.

3.2. Ethanolamine and phosphoethanolamine levels in serum and liver In our experiments, hepatocytes were not able to synthesis the necessary amounts of PE without a supply of Etn [2,4]. Ethanolamine comes off in the process of phospholipid synthesis from PE to phosphatidylserine in vivo. However, we are unable to ascertain whether it is sufficient to maintain all biofunctions. Possibly, it is supplied from several foods that have Etn, phosphoethanolamine, and PE. It has not been clarified whether serum Etn level is stable or not, and if Etn accumulates somewhere after eating a large quantity of Etn or PE. Serum Etn and phosphoethanolamine levels in the liver were higher in rats fed a high-cholesterol diet that included 2% egg yolk– derived PE for 2 weeks [22]. We examined Etn and phosphoethanolamine levels in the serum and liver. The serum Etn level was 25.0 F 1.2 lM in rats fed HF/HC diets and 31.3 F 1.1 lM in rats fed the same diet with Etncontaining water. Ethanolamine and phosphoethanolamine levels in the liver are shown in Fig. 2. The Etn level in the liver was the same in rats fed AIN-93M and HF/HC diets

µmol/g Liver

6.0 5.0

Phosphoethanolamine

4.0

Ethanolamine

3.0 2.0

b

b

ab a

a

a

1.0 0.0 AIN-93M

Fig. 3. Northern blot analysis on apoA-I, apoB, and apoE expression in liver (A) and small intestine (B) in rats fed an HF/HC diet with 1 mg/mL Etn or 1 mg/g Bf. mRNA at 5 lg per lane for apoB and tRNA and at 1 lg per lane for apoA-I, apoE, and GAPDH isolated from rat livers and small intestine were used for Northern blot analysis as described in Methods and materials.

and 1.5 times higher in rats given Etn than in those receiving only water. Phosphoethanolamine level in the liver was a little bit higher in rats fed AIN-93M than in those fed HF/ HC diets and 2.5 times higher in rats given Etn than in those receiving only water. These results showed that Etn levels in the serum and liver were very stable under homeostatic control and that much of the supplied Etn might be accumulated as phosphoethanolamine in the liver. 3.3. Northern blot analysis of apoA-I, apoB, and apoE in the liver and small intestine Ethanolamine and Bf decreased serum cholesterol levels, especially the secretion of VLDL cholesterol from the liver into the serum. The action of apolipoproteins, such as apoA-I, apoB, and apoE, has been investigated with respect to the regulation of serum lipid levels [18,19]. We examined the mRNA expression of apoA-I, apoB, and apoE in the liver and small intestine to determine whether they were related to cholesterol levels in the serum. As shown in Fig. 3, the mRNA expression of apoB was much lower (50%) in the liver of rats given Etn, but apoA-I in the liver and apoE in the small intestine were slightly higher, whereas apoE in the liver and apoB and apoA-I in the small intestine did not change. In the case of Bf, mRNA expressions of apoA-I and apoB were lower in the liver and small intestine of rats compared with the controls. In addition, the expression of apoE was higher in the small intestine. The decrease in apoB mRNA expression in the liver was the most significant change that occurred in rats given Etn and rats given Bf. GAPDH levels were almost identical for each diet group of rats. These results showed that the decrease of apoB mRNA expression might be related to the decrease of VLDL secretion from the liver.

+ Etn HF/HC diet

Fig. 2. Concentrations of Etn and phosphoethanolamine in liver in rats fed AIN-93M or an HF/HC diet with and without 1 mg/mL Etn. Values represent mean F SEM, n = 6. Values with different superscripts are significantly different with P b .05 by Fisher-PLSD.

4. Discussion In this study, we examined the effects of Etn in rats fed an HF/HC diet and compared them to that of Bf to investigate the relationship between serum lipid levels and the PEMT pathway. The results showed that Etn decreased

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serum cholesterol levels, especially VLDL and LDL cholesterol, in rats fed an HF/HC diet. The effect of Bf, which decreased cholesterol levels in the serum and liver, was greater than that of Etn when the rats were almost the same weight. Ethanolamine, a nutritional factor, is absorbed from the small intestine and reaches the liver by passing through the portal vein [23]. Absorbed Etn is used to synthesize PE and phosphatidylcholine, which are synthesized via the methylation of PE by PEMT in hepatocytes [4]. Ethanolamine and Bf inhibit the PEMT pathway and phosphatidylcholine synthesis and increase PE levels in vivo and in vitro [4,10,16]. Our results showed that phosphatidylcholine and PE levels are lower in liver microsomes of rats fed HF/HC diets than rats on a cholesterol-free diet (data not shown) because phosphatidylcholine is used to synthesize VLDL. The change of phospholipid composition might induce abnormalities in cell function. Phosphatidylcholine and PE levels were increased in rats that were fed a Bfcontaining diet and recovered nearly up to a normal level, but these levels were not increased in the rats given Etn. However, Etn and, especially, phosphoethanolamine accumulated in the liver. We postulate that Etn is used in PE and phosphatidylcholine synthesis and that the balance that is not used then accumulates as phosphoethanolamine in the liver. A higher concentration of Etn might be necessary to maintain appropriate levels of PE and phosphatidylcholine in hepatocytes. Previous research on Bf and PEMT found that Bf decreased the VLDL secretion only when active PE methylation was maintained with Etn in hepatocytes, and that apoB48 was then also decreased in secreted VLDL [17]. The secretion of VLDL, LDL, and apoB100 is decreased from hepatocytes derived from PEMT gene knockout mice compared with those from the wild-type [24]. Furthermore, serum cholesterol is lower and the amount of cholesterol esters and triacylglycerols in the liver were significantly higher in male mice that lacked PEMT and were fed an HF/HC diet than in the wild-type mice [25]. These results showed that the PEMT pathway might regulate cholesterol metabolism and, especially, the secretion of VLDL from the liver and decrease serum lipid and hepatic lipid levels via phosphatidylcholine and PE levels in cell membranes in rats fed an HF/HC diet. The difference in phosphatidylcholine and PE levels might cause different degrees of Etn and Bf inhibition of PEMT. Next, we examined the results of mRNA expression of apoA-I, apoB, and apoE. ApoA-I, apoB, and apoE are associated with chylomicrons, VLDL, and LDL particles, whereas apoA-I is associated with HDL particles, and they play an important role in cholesterol transport. Our results showed that Etn only suppressed apoB mRNA expression in the liver. In addition, apoA-I mRNA expression was unaffected in the liver and small intestine. These results are in agreement with the lower VLDL cholesterol level and the same level of HDL cholesterol. In addition, Bf

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suppressed apoA-I and apoB mRNA levels in the liver and small intestine and enhanced apoE mRNA expression in the small intestine of rats. These results are in agreement with the lower level of VLDL cholesterol, and the absorption of cholesterol from the small intestine might be reduced. Accordingly, only mRNA expression of apoB was drastically suppressed in the liver of rats given Etn and rats given Bf. There have been several reports related to apoB. Hepatic and intestinal apoB mRNA levels decreased in heterozygotes (apoB+/-), presumably contributing to decreased LDL levels through decreased synthesis of apoB-containing lipoproteins [19]. The rat liver synthesizes apoB-100 and apoB-48 [26], and high-fat and HF/HC diets increase apoB mRNA editing relative to apoB-100 mRNA in the liver [27]. The secretion of apoB is decreased by fasting, whereas apoB mRNA levels remain constant. Additional (posttranscription) mechanisms appear to play a role in regulating apoB secretion [28]. It is not certain from these reports whether the secretion of VLDL from the liver is regulated by the mRNA level of apoB, but the amount of apoB always decreases when VLDL secretion decreases. Taken together, these data suggest that the inhibition of PEMT regulated the change of the composition of phospholipids, although it remains unclear how and what regulates the synthesis of apoB. The inhibition of the PEMT pathway might regulate serum cholesterol level via the composition of phosphatidylcholine and PE and via the synthesis of apoB, especially apoB mRNA levels and/or apoB mRNA editing. These results showed that Etn decreased serum cholesterol, especially VLDL cholesterol and LDL cholesterol in rats fed an HF/HC diet and might regulate serum cholesterol levels via the expression of apoB mRNA in liver. Thus, Etn derived from several foods might be useful in regulating serum cholesterol level.

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