Comparative effects of short- and long-term feeding of safflower oil and perilla oil on lipid metabolism in rats

Comparative Biochemistry and Physiology Part B 121 (1998) 223 – 231

Comparative effects of short- and long-term feeding of safflower oil and perilla oil on lipid metabolism in rats Miyuki Ihara *, Hayato Umekawa, Takao Takahashi, Yukio Furuichi Laboratory of Nutritional Chemistry, Department of Agricultural Chemistry, Faculty of Bioresources, Mie Uni6ersity, Tsu, Mie 514 -8507, Japan Received 21 April 1998; received in revised form 22 June 1998; accepted 13 August 1998

Abstract Diets high in linoleic acid (20% safflower oil contained 77.3% linoleic acid, SO-diet) and a-linolenic acid (20% perilla oil contained 58.4% a-linolenic acid, PO-diet) were fed to rats for 3, 7, 20, and 50 days, and effects of the diets on lipid metabolism were compared. Levels of serum total cholesterol and phospholipids in the rats fed the PO-diet were markedly lower than those fed the SO-diet after the seventh day. In serum and hepatic phosphatidylcholine and phosphatidylethanolamine, the proportion of n-3 fatty acids showed a greater increase in the PO group than in the SO group in the respective feeding-term. At the third and seventh days after the commencement of feeding the experimental diets, expressions of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase mRNA were significantly higher in the SO group than those in the PO group, although the difference was not observed in the longer term. There were no significant differences in the LDL receptor mRNA levels between the two groups through the experimental term, except 3-days feeding. These results indicate that a-linolenic acid has a more potent serum cholesterol-lowering ability than linoleic acid both in short and long feeding-terms. © 1998 Elsevier Science Inc. All rights reserved. Keywords: Rat; Safflower oil; Perilla oil; Fatty acids; Cholesterol; Feeding-term; HMG-CoA reductase; mRNA expression

1. Introduction Dietary fatty acids are known to affect serum cholesterol concentration and play an important role in the development of atherosclerotic disease [3,14,25,27]. Diet rich in saturated fatty acids (SFA) increases serum lipid concentration, while polyunsaturated fatty acids (PUFA) decrease it [34]. Mensink and Katan [30] and Hegsted et al. [18] also showed that SFA raises, and PUFA lowers, total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C). Two types of PUFA Abbre6iations: ALA, a-linolenic acid; HDL, high-density lipoprotein; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; LA, linoleic acid; LDL, low-density lipoprotein; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PL, phospholipids; PO, perilla oil; SO, safflower oil; TC, total cholesterol; TG, triglycerides. * Corresponding author. Tel.: + 81 59 2319612; fax: + 81 59 2319684; e-mail: [email protected]

occur in diets, n-6 and n-3 polyunsaturates. Linoleic acid (LA, 18:2 n-6), the parent molecule of n-6 PUFAs, was a well-known hypocholesterolemic fatty acid [14]. Garg et al. [13] observed a slight decrease in plasma cholesterol by feeding rats safflower oil (SO) (rich in LA) for 4 weeks as compared with beef tallow (rich in SFA), but this decrease was accompanied by increase in hepatic cholesterol content. Moreover, plasma cholesterol levels were higher in rodents fed animal fats as compared with high-linoleate vegetable oils in a short feeding period (2 weeks), but the difference became smaller after longer feeding period (6 weeks) [37]. In the n-3 PUFA family, a-linolenic acid (ALA, 18:3 n-3) serves as a precursor for biosynthesis of eicosapentaenoic acid (EPA, 20:5 n-3) and docosahexaenoic acid (DHA, 22:6 n-3). It was shown that n-3 PUFAs decrease serum TC in human and experimental animals [16,17]. On the other hand, high-linoleate vegetable oils

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are not hypocholesterolemic as compared with high-alinolenate vegetable oils in aged mice [24,40], rats [40], and adult humans [45] after long-term feeding. LDL receptor participates in the rate-determining steps in lipoprotein uptake by cells [28]. Dietary fatty acids regulate plasma LDL levels primarily by affecting receptor-mediated LDL transport, and this regulation is displayed largely at the mRNA level [21,22]. The expression of LDL receptor exerts a strong and independent positive effect in rats fed PUFA diet than those fed SFA diet [32,34]. Moreover, mRNA abundances of LDL receptor and 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase, the latter catalyzes the formation of mevalonate from HMG-CoA and is thought to be a rate-limiting enzyme for cholesterol synthesis in liver, are generally thought to be regulated in the same direction if not to the same degree [31]. El-Sohemy and Archer [10] showed that HMGCoA reductase activity is significantly lower in the hepatic microsomes of rats fed n-3 PUFA-diet than in those fed n-6 PUFA-diet. In the present investigation, we examined the effects of short- and long-term feedings of SO and perilla oil (PO) on lipid metabolism and fatty acid compositions of serum and tissue lipids in rats. The present study also compared the effects of SO and PO, and feedingterms on the mRNA expressions of hepatic HMGCoA reductase, LDL receptor, and cholesterol 7a-hydroxylase.

2. Materials and methods

2.1. Animals and diets Male Wistar rats, 3-weeks old, were purchased from SLC (Shizuoka, Japan) and housed individually in stainless steel cages with wire bottoms in the room controlled for temperature (209 2°C), and 12 h-light/ dark cycle (light: 8:00 – 20:00 h). Dietary fat was made to 20% (wt/wt) in each diet with SO (SO-diet) (Rinoru Oil Mills, Nagoya, Japan) or PO (PO-diet) (Ohta Oil, Okazaki, Japan). The fatty acid compositions of these oils are shown in Table 1. The experimental diets (Table 2) were made according to the formula recommended by American Institute of Nutrition [38]. AIN93 mineral and vitamin mixtures [38] were purchased from Oriental Yeast (Tokyo, Japan). After the rats were acclimated to the facility on a laboratory chow (Type MF, Oriental Yeast) for 1 week, they were divided into nine groups of five animals each and were given the experimental diets ad libitum for 3, 7, 20, and 50 days. Drinking water was also supplied freely. On the last day of each feedingterm, the rats were deprived of the food for 4 h (9:00 –13:00 h) and blood was withdrawn from the

Table 1 Fatty acid compositions of dietary fats (%) Fatty acid

SO

PO

16:0 18:0 18:1 (n-9) 18:2 (n-6) 18.3 (n-3) 20:0 22:0 n-3/n-6

6.7 2.5 12.7 77.3 0.4 0.2 0.2 5.2×10−3

6.2 1.4 18.5 15.5 58.4 ND ND 3.8

SO, safflower oil; PO, perilla oil; ND, not detected.

abdominal aorta under diethyl ether anesthesia. The blood was centrifuged at 1700× g for 15 min to separate serum. The liver and perirenal adipose tissue were excised immediately and frozen at − 80°C for Northern blot analysis of mRNA and − 30°C for lipid analysis. The animal experiments were conducted in accordance with the guidelines of the Committee on Animal Research of our university.

2.2. Lipid analyses Total hepatic lipids were extracted according to the method of Folch et al. [11]. Serum and hepatic lipids were analyzed for TC, triglycerides (TG), phospholipids (PL) and high-density lipoprotein cholesterol (HDL-C), using commercial kits (‘Cholesterol C2 Test-Wako’ for TC, ‘Triglyceride Test-Wako’ for TG, and ‘Phospholipid B Test-Wako’ for PL from Wako Pure Chemical Industries, Osaka, Japan, and ‘HDLC2 Daiichi’ for HDL-C from Daiichi Pure Chemicals, Tokyo, Japan). Hepatic total lipids (TL) were determined after the Folch extraction gravimetrically. All the organic solvents contained 0.01% butylated hydroxytoluene [46]. Table 2 Compositions of the experimental diets (%) Ingredients

SO-diet

PO-diet

Casein Safflower oil Perilla oil Mineral mixturea Cellulose Vitamin mixturea DL-Methionine Choline chloride Cornstarch Sucrose

20.0 20.0 — 3.5 5.0 1.0 0.3 0.2 15.0 to 100

20.0 — 20.0 3.5 5.0 1.0 0.3 0.2 15.0 to 100

a

AIN-93 [38].

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Table 3 Body growth and food intake in rats fed safflower and perilla oils Feeding-term (days)

Diet

Initial body weight (g) 93.4 93.7

0 3

SO PO

96.0 93.1 97.4 92.0

7

SO PO

20 50

Weight gain (g) —

Food intake (g) —

Liver weight (g 100g−1 body weight) 4.4 90.1

17.0 9 0.3 13.6 9 0.9**

37.7 90.5 34.4 90.9*

4.2 90.1 4.8 90.1**

101.093.9 101.692.5

41.6 91.8 39.8 92.0

81.6 9 3.0 78.5 91.9

4.5 90.2 4.8 9 0.1

SO PO

100.893.6 102.093.5

132.09 3.5 132.2 96.6

285.5 94.6 274.3 9 9.7

4.3 9 0.0 5.1 9 0.4*

SO PO

101.2 94.2 100.4 93.9

278.2 910.3 274.49 15.6

798.6 9 27.5 830.3 936.9

3.8 90.2 3.3 90.1*

Values are means 9 S.E. of five rats/group. The values for 0 day represent the initial levels. SO, safflower; PO, perilla oil. * Significantly different from the corresponding SO group (*PB0.05, **PB0.01).

2.3. cDNA probes The rat LDL receptor cDNA probe was a gift from Dr F Horio (Nagoya University, Japan). HMG-CoA reductase and cholesterol 7a-hydroxylase cDNA probes were synthesized from the rat hepatic total RNA by RT-PCR with sense primer: 5%-ACAATGTTGTCAAGACTTTT-3% and antisense primer: 5%CCTCCTATGCTACCAGCCAT-3% corresponding to the positions 104–123 and 2327 – 2346 of rat HMGCoA reductase cDNA [15], and with sense primer: 5%-TTGATTCCGTACCTGGGCTGTGCTC-3% and antisense primer: 5%-AGTGAAGTCCTCCTTAGCTGTGCGG-3% corresponding to the positions 147–171 and 1124–1148 of rat cholesterol 7a-hydroxylase cDNA [35], respectively. Amplified fragment of each HMG-CoA reductase and cholesterol 7a-hydroxylase cDNA was subcloned into pCR™2.1 plasmid vector (Invitrogen, San Diego, CA) and the both strands were sequenced by the dideoxyribonucleotide chain termination method [41].

2.4. Northern blot analysis Total RNA was extracted using guanidinium-thiocyanate with acid phenol-chloroform according to the method described by Chomczynski and Sacchi [4]. Total RNA (20 mg) from rat liver was separated by electrophoresis in a 1% agarose gel containing 2.2 M formaldehyde and transferred to a nylon membrane (NYTRAN, Schleiche and Schuell). The membrane was prehybridized with salmon sperm DNA (200 mg ml − 1) at 42°C for 2 h and then separately hybridized with the radiolabeled rat HMG-CoA reductase, LDL receptor, cholesterol 7a-hydroxylase and glyceraldehyde-3-phosphate dehydrogenase (G3PDH) cDNA probe (TOYOBO, Japan), as an internal control, in

50% formamide, 5× SSPE (1× SSPE: 0.18 M NaCl, 10 mM NaH2PO4 and 1 mM EDTA, pH 7.4) and 0.1% SDS at 42°C for 16 h and followed by sequential washing with 2× SSC (1× SSC: 0.15 M NaCl and 15 mM sodium citrate) containing 0.1% SDS at 65°C for 30 min, 1× SSC containing 0.1% SDS at 65°C for 30 min, and 0.5 × SSC containing 0.1% SDS at 65°C for 30 min. After washing, the membrane was subjected to autoradiography. The amount of the each HMG-CoA reductase, LDL receptor, cholesterol 7a-hydroxylase, and G3PDH mRNA hybridized with the corresponding probe was analyzed by a Bio Imaging Analyzer BAS 1000 system (Fuji Photo, Tokyo, Japan).

2.5. Fatty acid analysis Lipids were extracted from serum or homogenized tissue samples according to the method of Folch et al. [11] and the extracts were evaporated to dryness under nitrogen. Phosphatidylcholine (PC), phosphatidylethanolamine (PE) and TG were separated by two-dimensional Silica Gel (Merck 60) thin-layer chromatography using petroleum ether/diethyl ether/ acetic acid (82:18:1, v/v/v) and chloroform/methanol/ water (65:27:1, v/v/v) as the first and second developing solvents, respectively [29]. The PC, PE, and TG fractions were extracted from silica gel on the plate and transmethylated with H2SO4-methanol and fatty acid methyl esters were analyzed by gas-liquid chromatography (GC-14B) and data processor (Chromato Pac C-R7A) from Shimadzu. (Kyoto, Japan) equipped with a 30 m×0.32 mm (i.d.) HR-SS-10 column (Shinwa, Japan), and a flame-ionization detector [23]. Fatty acids were identified based on the relation between the carbon number of a homologue and retention time or by comparing the retention time with that of a reference standard.

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Fig. 1. Effects of safflower and perilla oils on serum lipid levels in rats. Values are means 9S.E. of five rats/group. a, b, c Different letters are assigned when means differ from the 0 day significantly at PB 0.05. * Significantly different from the corresponding SO group (*PB 0.05, **PB 0.01). TC, total cholesterol; HDL-C, high-density lipoprotein cholesterol; PL, phospholipids; TG, triglycerides.

2.6. Statistical analysis

3.2. Serum lipids

The results are presented as the mean9 S.E. for five animals. The data were analyzed by means of analysis of variance, using the Microsoft excel 5.0 (Microsoft cooperation). Differences between means were assessed with the Student’s t-test [43].

The serum TC (Fig. 1A) and PL (Fig. 1D) levels were significantly lower in the PO than the SO group after the seventh day. In the PO group, TC level decreased remarkably during the first 7 days ( − 32%, PB0.01), whereas the SO-diet kept the initial level (Fig. 1A). The PO group kept lower values in HDL-C than the SO group through the experimental period with a significant difference at the 20th day (Fig. 1B). The TG level was significantly lower in the PO than the SO group during the first 7 days (Fig. 1C) with a remarkable difference at the seventh day. The PO group kept significantly lower values in PL than the SO group after the seventh day (Fig. 1D).

3. Results In the present study, the values for animal growth, lipid component, fatty acid composition, and mRNA expression at the zero day (0 day), when rats had been given a commercial chow for 7 days, were employed as the initial levels.

3.1. Growth parameters

3.3. Hepatic lipids

Animal growth and liver weights are shown in Table 3. No significant differences were observed in the weight gain and food intake between the two dietary groups during the respective feeding-term, except 3days feeding. The liver weights of the rats fed the PO-diet were significantly higher than those fed the SO-diet at the third and 20th days. However, the weight was significantly lower in the PO- than the SO-diet at the 50th day.

The effects of feeding-term on the hepatic lipid levels are shown in Fig. 2. The hepatic cholesterol levels in both the SO and PO groups showed their maxima at the seventh day (Fig. 2A). A significantly lower level of cholesterol was observed in the PO than the SO group at the 50th day (PB 0.05). The PL levels of both groups showed nearly the same time course (Fig. 2B). The TG levels of both groups increased markedly (about 4-fold increase) during the first 7 days, although

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Fig. 2. Effects of safflower and perilla oils on hepatic lipid levels in rats. Values are means 9 S.E. of five rats/group. a, b, c Different letters are assigned when means differ from the 0 day significantly at P B0.05. * Significantly different from the corresponding SO group (*PB 0.05, **PB 0.01). TL, total lipids; Chol, hepatic cholesterol; PL, phospholipids; TG, triglycerides.

the PO group showed a significantly lower level at the 50th day (Fig. 2C). Both the SO and PO groups showed similar time courses in the TL level (Fig. 2D).

3.4. Fatty acid compositions of serum and tissue lipids The fatty acid compositions of hepatic PC are shown in Table 4. The SO group decreased gradually in the percent of 22:6 n-3. In the PO group, n-3 PUFAs (18:3 and 20:5) were found as early as 3 days after the commercial laboratory chow was replaced by the experimental diet, and did not increase or decrease thereafter. The PO group showed higher proportions in 18:1 n-9 and 18:2 n-6 than the SO group after the third day. However, the percent of 20:4 n-6 in the PO group was remarkably lower than that of the SO group through the experimental period. The PO-diet showed a significant decrease in 20:4 n-6 level as compared to the SO-diet in the first 3 days. The time-dependent changes in the fatty acid composition of serum PC and hepatic PE were generally similar to that of hepatic PC (data not shown). Fatty acid profiles of TGs of serum, perirenal adipose tissue lipids, and liver reflected well the types of dietary fats (data not shown).

3.5. Response of HMG-CoA reductase mRNA gene expression to dietary oil and feeding-term A representative autoradiogram depicting the changes in hepatic HMG-CoA reductase and G3PDH mRNA expressions is shown in Fig. 3, and HMGCoA reductase mRNA levels are summarized in Fig. 4. The HMG-CoA reductase mRNA levels in the SO group were lowered in the first 3 days, and then they returned to the 0 day level and finally increased to a higher level at the 50th day. The levels in the rats fed the PO-diet were decreased remarkably in the first 3 days, followed by a gradual elevation with feedingterm. The mRNA levels were significantly lower in the PO group compared with the SO group in the first 7 days and the significant difference between the two groups disappeared after the 20th day.

3.6. Hepatic LDL receptor and cholesterol 7a-hydroxylase mRNA le6els The effects of dietary fat and feeding-term on the hepatic LDL receptor and cholesterol 7a-hydroxylase mRNA levels are shown in Fig. 5. The LDL receptor mRNA levels in both diets remained stable during the first 7 days and approximately tripled at the 50th

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Table 4 Fatty acid compositions of hepatic phosphatidylcholine (%) Fatty acid

0 day

3 days

7 days

20 days

50 days

0.1 90.0c 0.1 90.0c

0.2 9 0.0b 0.1 90.0c

16.6 9 0.7b 14.7 9 0.5b

14.9 90.6b 15.1 9 0.3b

14.6 9 0.3c 15.4 9 0.8b

0.19 0.0a 0.39 0.0a*

0.2 90.0a 0.7 90.0b

0.2 90.0a B0.1

0.2 90.1a B0.1

27.590.9b 23.991.0a*

21.3 9 0.3c 24.190.8a*

26.2 9 1.3b 23.3 9 0.7a

28.3 9 1.4b 26.8 9 0.8b

14:0

0.29 0.0a

0.29 0.0b 0.29 0.0b

16:0

19.6 9 0.3a

17.790.5b 16.290.8b

16:1

0.39 0.2a

18:0

23.6 9 0.6a

0.2 90.0b 0.1 90.0c*

18:1(n-9)

7.49 0.2a

5.49 0.3b 9.89 0.4b**

5.3 9 0.2b 8.8 90.1b**

4.6 90.5b 6.5 90.2c**

4.6 90.0b 5.8 9 0.2c*

18:2(n-6)

12.59 0.4a

12.290.5a 15.990.4b**

14.0 9 0.1b 16.4 9 0.3b**

10.9 9 0.6a 16.5 90.4b**

8.7 90.6c 14.9 90.6c**

18:3(n-6)

0.29 0.1a

0.39 0.1a 0.39 0.1a

0.29 0.0a ND

B0.1 B0.1

0.1 90.1b 0.2 90.1a

20:3(n-6)

0.49 0.1a

1.09 0.1b ND

1.4 90.2b ND

0.4 90.2a ND

1.0 90.2b ND

20:4(n-6)

27.69 0.3a

33.091.1b 16.29 1.5b**

35.7 90.5b 15.2 90.6b**

41.6 9 0.6c 17.4 90.4b**

40.9 9 0.7c 18.3 9 0.8c**

22:4(n-6)

ND

ND ND

0.5 9 0.2a ND

2.6 90.1b ND

3.19 0.1c ND

18:3(n-3)

ND

ND 1.59 0.2a

ND 1.9 90.2a

ND 1.3 90.1a

ND 0.9 9 0.1a

18:4(n-3)

0.49 0.1a

ND 0.49 0.0a

ND 0.8 90.0b

ND 1.2 90.1c

ND 1.5 90.1c

20:5(n-3)

ND

ND 9.59 1.1a

ND 10.3 9 1.0a

ND 11.6 9 1.0a

ND 9.1 90.6a

22:5(n-3)

ND

ND ND

ND ND

ND 0.2 90.0a

ND 0.2 90.1a

22:6(n-3)

7.79 0.2a

4.59 0.2b 5.69 0.8b

4.5 90.4b 7.0 90.7a*

2.6 90.4c 6.3 90.4c**

0.9 9 0.2c 6.2 90.4c**

Values are means9S.E. of five rats/group. The values for 0 day represent the initial levels. Upper and lower rows for each fatty acid represent time-dependent changes in proportions of the fatty acid on rats fed the SO- and PO-diets, respectively. ND, not detected; SO, safflower oil; PO, perilla oil. a, b, c Different superscript letters are assigned when means differ significantly at PB0.05. * Significantly different from the SO group (*PB0.05, **PB0.01).

day. However, no diet-related difference between the two groups was observed through the experimental period, except at the third day. The cholesterol 7a-hydroxylase mRNA levels at the third and 20th days were higher in the PO than in the SO group, but the differences were not statistically significant. On the other hand, the level was significantly lower at the seventh and 50th days in the PO than in the SO group.

4. Discussion In this study, we used 4-week old rats and fed them the experimental diets for 3 – 50 days. It may be esti-

mated that the rats finish their growth at about 80 days after birth. Accordingly, in this investigation, a longest feeding-term of 50 days (roughly one twentieth of the life span) was adopted to evaluate the consequences of long-term feedings. The present results suggest that the types of fatty acids, 18:2 n-6 and 18:3 n-3, and feedingterm behave differently in affecting serum and hepatic lipid metabolism and fatty acid compositions of tissue lipids. The rats fed the SO-diet grew faster than those fed the PO-diet in the first 3 days (Table 3). This may be explained partly by the finding that the food consumption in the first 3 days was somewhat higher in the SO group. However, there were no significant differences in food intake between the two dietary groups after the seventh day. Accordingly, the changes ob-

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Fig. 3. Hepatic HMG-CoA reductase mRNA expressions. HMGCoA, 3-hydroxy-3-methylglutaryl coenzyme A; G3PDH, glyceraldehyde-3-phosphate dehydrogenase; SO, safflower oil; PO, perilla oil.

served in cholesterol metabolism and fatty acid composition of tissue lipids may be considered to be due to the dietary-fat treatment and do not stem from the differences in the growth rate. Rapid adaptive changes in the serum TC and PL levels appear to occur within the first 7 days (shortterm), followed by nearly plateau levels from the seventh to the 50th day (long-term) in rats fed the PO-diet, although in the SO-diet the two lipid components kept their initial levels (Fig. 1A and Fig. 1D). Ishihara et al. [24] reported that an increased intake of high-linoleate vegetable oil is not useful for the prevention of hypercholesterolemia-associated disease, although dietary oils rich in n-3 fatty acids are useful. Thus, it may be emphasized that high-linoleate vegetable oils are not hypocholesterolemic as compared with high-a-linolenate ones in rats on both short- and long-term feedings. In this regard, it is noteworthy that increasing a-linole-

Fig. 4. Hepatic HMG-CoA reductase mRNA levels. Quantitation of HMG-CoA reductase mRNA levels was done as described in ‘materials and methods’. The figure depicts the level of each mRNA (mean9S.E., n =5) normalized to G3PDH mRNA content. c Significantly different from the 0 day level at PB 0.05. * Significantly different from the corresponding SO group at PB 0.05.

Fig. 5. Hepatic LDL receptor and cholesterol 7a-hydroxylase mRNA levels. Quantitation of LDL receptor (upper) and cholesterol 7a-hydroxylase (lower) mRNA levels was done as described in ‘materials and methods’. The figures depict the level of each mRNA (mean9 S.E., n =5) normalized to G3PDH mRNA content. c Significantly different from the 0 day level at PB0.05. * Significantly different from the corresponding SO group at P B0.05.

nate but decreasing linoleate has been proved to be effective for the prevention of atherosclerotic disease in humans [9]. The hepatic cholesterol contents in rats fed the POdiet, with statistical differences at the third and the 50th days, were lower than those in the SO group (Fig. 2A). This observation coincides with the finding of Garg et al. [13] who reported that a diet containing SO shifted cholesterol from plasma to liver pools, but a linseed oil diet (rich in a-linolenic acid) did not. Decreases in serum cholesterol level often accompany hepatic cholesterol accumulation [12,44,45]. In the present study, hepatic cholesterol level was significantly lower in the rats fed the PO-diet in comparison with the rats fed the SO-diet (Fig. 2A). Accordingly, it seems important to evaluate the consequences of relatively long-term administration of dietary fats. Consumption of the SO-diet resulted in higher levels of 20:4 n-6 of serum and hepatic PCs and hepatic PE than that of the PO-diet through the experimental term (Table 4). Higher proportions of the desaturated-chain elongated products of 18:3 n-3, i.e. 20:5 n-3 and 22:6

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n-3, were observed in hepatic PC and PE of rats fed the PO-diet in the longer feeding-term. In the liver, it is generally accepted that linoleic acid and a-linolenic acid are subjected to the same enzyme systems, desaturation and chain elongation, to form C20, C22 n-6 and longer n-3 fatty acids [3,8,20,42]. However, the conversion of a-linolenic acid to higher and more unsaturated metabolites was greater than that of linoleic acid due to a higher affinity of the rate-limiting enzyme, delta-6-desaturase, for the n-3 than for the n-6 essential fatty acids [2,47]. Lower proportion of monounsaturated fatty acids (18:1 n-9) was observed in both serum and hepatic PCs on the SO- as compared to the PO-diet. In this connection, a higher delta-9-desaturase activity in rats fed a-linolenic acid than in those fed linoleic acid was reported by Christiansen [5]. A number of studies have investigated the effects of dietary fats on lipoprotein metabolism in the rats [1,19,26,33,36]. Certain species of fatty acids, notably PUFA, have been shown to regulate specifically the expression of several genes [6,7]. Determinations of hepatic HMG-CoA reductase, LDL receptor, and cholesterol 7a-hydroxylase mRNA levels using Northern hybridization allow a speculation on potential of cholesterol-lowering mechanisms by n-3 and n-6 fatty acids. We found that suppression of HMG-CoA reductase mRNA levels may account indirectly for decreased cholesterol levels in serum of rats fed the PO-diet in the short-term feeding. In light of the present observations, fatty acids may play a direct role in regulating HMGCoA reductase gene expression even in a short feedingterm. The rats fed the PO-diet showed a higher level of LDL receptor mRNA as compared to those fed the SO-diet at the third day, although the level was the same as the initial level. However, there were no significant differences in the mRNA level between the two groups after the seventh day. In this regard, feeding n-3 fatty acids was reported to either decrease [39] or increase [48] the activity of the LDL receptor in rats. Present results show that mRNA expression of LDL receptor increases with age. In this study, lower expression of cholesterol 7a-hydroxylase mRNA was shown in the PO- than in the SO-diet at the 50th day, although higher expression of the mRNA is known to be effective for lowering serum cholesterol level. In this regard, in the PO-diet, lower expression of HMG-CoA reductase mRNA may contribute to lowering serum cholesterol level. As already mentioned, however, on the 50th day, serum cholesterol level was significantly lower in the PO than the SO group, although no difference was observed in the hepatic HMG-CoA reductase mRNA expression between the two groups. The reason for this discrepancy is not certain. Enzymatic activity of HMG-CoA reductase must be assayed in further experiments.

In conclusion, our present results have demonstrated that a-linolenic acid plays an important role in a regulation of serum cholesterol and the magnitude of regulation is more powerful as compared to linoleic acid. Dietary fats affect markedly fatty acid profiles of serum and hepatic PC and PE even in a short feeding-period. One of the mechanisms for lowering serum cholesterol by a-linolenic acid is considered to suppress the expression of mRNA for HMG-CoA reductase, a key enzyme responsible for cholesterol biosynthesis.

Acknowledgements We are very grateful to Dr Fumihiko Horio (Laboratory of Nutritional Biochemistry, Faculty of Agriculture, Nagoya University, Japan) and Dr Takeshi Ohkubo (Center for Molecular Biology and Genetics, Mie University, Japan) for their technical assistance and discussions. We thank Dr Ikuo Ikeda (Laboratory of Nutrition Chemistry, Faculty of Agriculture, Kyushu University, Japan) for TLC method. We also thank Rinoru Oil Mills and Ohta Oil for the generous gifts of safflower oil and perilla oil, respectively.

References [1] Balasubramaniam S, Simons LA, Chang S, Hickie JB. Reduction in plasma cholesterol and increase in biliary cholesterol by a diet rich in n-3 fatty acids in the rat. J Lipid Res 1985;26:684–9. [2] Be´zard J, Blond JP, Bernard A, Clouet P. The metabolism and availability of essential fatty acids in animal and human tissues. Reprod Nutr Dev 1994;34:539 – 68. [3] Caggiula AW, Mustad VA. Effects of dietary fat and fatty acids on coronary artery disease risk and total and lipoprotein cholesterol concentrations: epidemiologic studies. Am J Clin Nutr 1997;65:1597S– 610S. [4] Chomczynski P, Sacchi N. Single-step method for RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987;162:156 – 9. [5] Christiansen EN, Lund JS, Rørtveit T, Rustan AC. Effect of dietary n-3 and n-6 fatty acids on fatty acid desaturation in rat liver. Biochim Biophys Acta 1991;1082:57 – 62. [6] Clarke SD, Araham S. Gene expression: nutrient control of preand post-transcriptional events. FASEB J 1992;6:3146 –52. [7] Clarke SD, Jump DB. Regulation of gene transcription by polyunsaturated fatty acids. Prog Lipid Res 1993;32:139–49. [8] Cook HW, Spence MW. Interaction of (n-3) and (n-6) fatty acids in desaturation and chain elongation of essential fatty acids in cultured glioma cells. Lipids 1987;22:613 – 9. [9] de Lorgeril M, Renand S, Mamelle N, Salen P, Martin J-L, Monjaud I, Guidollet J, Touboul P, Delaye J. Mediterranean alpha-linolenic acid-rich diet in secondary prevention of coronary heart disease. Lancet 1994;343:1454 – 9. [10] El-Sohemy A, Archer MC. Regulation of mevalonate synthesis in rat mammary glands by dietary n-3 and n-6 polyunsaturated fatty acids. Cancer Res 1997;57:3685 – 7. [11] Folch J, Lees M, Sloane-Stanley GH. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem 1957;226:497 – 509.

M. Ihara et al. / Comparati6e Biochemistry and Physiology, Part B 121 (1998) 223–231 [12] Fukushima M, Akiba S, Nakano M. Comparative hypocholesterolemic effects of six vegetable oils in cholesterol-fed rat. Lipids 1996;31:415 – 9. [13] Garg ML, Sebokova E, Wierzbicki A, Thomson ABR, Clandinin MT. Differential effects of dietary linoleic and a-linolenic acid on lipid metabolism in rat tissues. Lipids 1988;23:847 – 52. [14] Grundy SM, Denke MA. Dietary influences on serum lipids and lipoproteins. J Lipid Res 1990;31:1149–72. [15] Hangjiong, C, Williams, D, Shapiro, D. Cloning and sequencing of rat 3-hydroxy-3-methylglutaryl coenzyme A reductase. The nucleotide sequence has been revealed in the EMBL/GenBank/ DDBJ Data Bank with accession number M29249;1989. [16] Harris WS. n-3 Fatty acids and serum lipoproteins: animal studies. Am J Clin Nutr 1997;65:1611S–16S. [17] Harris WS. n-3 Fatty acids and serum lipoproteins: human studies. Am J Clin Nutr 1997;65:1645S–54S. [18] Hegsted DM, Ausman LM, Johnson JA, Dallal GE. Dietary fat and serum lipids: an evaluation of the experimental data. Am J Clin Nutr 1993;57:875–83. [19] Hirai K, Ohbo Y, Nakano T, Izutani K. Effects of dietary fats and phytosterol on serum fatty acid composition and lipoprotein cholesterol in rats. J Nutr Sci Vitaminol 1984;30:101–12. [20] Horrobin DF. Nutritional and medical importance of gammalinolenic acid. Prog Lipid Res 1992;31:163–94. [21] Horton JD, Cuthbert JA, Spady DK. Dietary fatty acids regulate hepatic low density lipoprotein (LDL) transport by altering LDL receptor protein and mRNA levels. J Clin Invest 1993;92:743 – 9. [22] Horton JD, Cuthbert JA, Spady DK. Regulation of hepatic 7a-hydroxylase expression by dietary psyllium in the hamster. J Clin Invest 1994;93:2084–92. [23] Ikeda I, Tomari Y, Sugano M. Interrelated effects of dietary fiber and fat on lymphatic cholesterol and triglyceride absorption in rats. J Nutr 1980;119:1383–7. [24] Ishihara A, Ito A, Sakai K, Watanabe S, Kobayashi T, Okuyama H. Dietary high-linoleate safflower oil is not hypocholesterolemic in aged mice after a long-term feeding-comparison with lard, perilla oil and fish oil. Biol Pharm Bull 1995;18:485 – 90. [25] Khosla P, Sundram K. Effects of dietary fatty acid composition on plasma cholesterol. Prog Lipid Res 1996;35:93–132. [26] Kris-Etherton PM, Ho CY, Fosmire MA. The effect of dietary fat saturation on plasma and hepatic lipoproteins in the rat. J Nutr 1984;114:1675 –82. [27] Kris-Etherton PM, Yu S. Individual fatty acid effects on plasma lipids and lipoproteins: human studies. Am J Clin Nutr 1997;65:1628S– 44S. [28] Lestavel S, Fruchart JC. Lipoprotein receptors. Cell Mol Biol 1994;40:461 – 81. [29] Mangolo HK. Thin-layer chromatography of lipids. J Am Oil Chem Soc 1964;41:762–73. [30] Mensink RP, Katan MB. Effects of dietary fatty acids on serum lipids and lipoproteins: a meta-analysis of 27 trials. Arterioscler Thromb 1992;12:911–9. [31] Molowa DT, Cimis GM. Co-ordinate regulation of low-densitylipoprotein receptor and 3-hydroxy-3-methylglutaryl-CoA reduc-

.

[32]

[33] [34]

[35]

[36]

[37]

[38]

[39]

[40]

[41] [42] [43] [44] [45]

[46]

[47]

[48]

231

tase and synthase gene expression in HepG2 cells. Biochem J 1989;260:731 – 6. Mustad VA, Ellsworth JL, Cooper AD, Kris-Etherton PM, Etherton TD. Dietary linoleic acid increases and palmitic acid decreases hepatic LDL receptor protein and mRNA abundance in young pigs. J Lipid Res 1996;37:2310 – 23. Nestel PJ. Dietary factors affecting lipoprotein metabolism. Adv Exp Med Biol 1985;183:253 – 63. Nicolosi RJ. Dietary fat saturation effects on low-density lipoprotein concentrations and metabolism in various animal models. Am J Clin Nutr 1997;65:1617S– 27S. Noshiro M, Nishimoto M, Okuda K. Rat liver cholesterol 7a-hydroxylase: pretranslational regulation for circadian rhythm. J Biol Chem 1990;265:10036– 41. Osada J, Fernandez SA, Diaz MJ, Miro OM, Debrian JA, Carrizosa C, Ordovas JM, Palacios AE. Differential effect of dietary fat saturation and cholesterol on hepatic apolipoprotein gene expression in rats. Atherosclerosis 1994;108:83 – 90. Otto DA, Tsai CE, Baltzell JK, Wooten JT. Apparent inhibition of hepatic triacylglycerol secretion, independent of synthesis, in high-fat fish oil-fed rats: role for insulin. Biochim Biophys Acta 1991;1082:37 – 48. Reeves PG, Nielsen FH, Fahey GC. AIN-93 purified diets for laboratory rodents: final report of the American Institute of Nutrition ad hoc writing committee on the reformulation of the AIN-76A rodent diet. J Nutr 1993;123:1939 – 51. Roach PD, Kambouris AM, Trimble RP, Topping DL, Nestel PJ. The effect of dietary fish oil on hepatic HDL and LDL receptor activities in the rat. FEBS Lett 1987;222:159 –62. Sakai K, Shimokawa T, Kobayashi T, Okuyama H. Lipid lowering effects of high linoleate and high-a-linolenate diets in rats and mice: consequence of long-term feedings. Chem Pharm Bull 1992;40:2129 – 32. Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain terminating inhibitors. Proc Natl Acad Sci USA 1977;74:5463–7. Simopoulos AP. Omega-3 fatty acids in health and disease and in growth and development. Am J Clin Nutr 1991;54:438–63. Snedecor, GW, Cochran, WG. Statistical Methods, 6th ed. Ames: Iowa State University Press, 1976, pp 472 – 503. Sprecher M. Biochemistry of essential fatty acids. Prog Lipid Res 1981;20:13 – 22. Strandberg TE, Salomaa VV, Naukkarinen VA, Vanhanen HT, Sarna SJ, Miettinen TA. Long-term mortality after 5-year multifactorial primary prevention of cardiovascular diseases in middle-aged men. J Am Med Assoc 1991;266:1225– 9. Takamura H, Narita H, Park HJ, Tanaka K, Matsuura T, Kito M. Differential hydrolysis of phospholipid molecular species during activation of human platelets with thrombin and collagen. J Biol Chem 1987;262:2262 – 9. Ulmann L, Blond JP, Maniongui C, Poisson JP, Durand G, Be´zard J, Pascal G. Effect of age and dietary essential fatty acids on desaturase activities and on fatty acid composition of liver microsomal phospholipids of adult rats. Lipids 1991;26:127–33. Ventura MA, Woollett LA, Spady DK. Dietary fish oil stimulates hepatic low density lipoprotein transport in the rat. J Clin Invest 1989;84:528 – 37.