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Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, Shizuoka 422-8529, Japan
1To whom correspondence should be addressed. E-mail: acksugi{at}agr.shizuoka.ac.jp.
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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5-, 6- and 9-desaturases in liver microsomes were significantly decreased by eritadenine and ethanolamine; there was a significant correlation between the activity of 5- or 6-desaturase and the proportion of PE in the total phospholipids or the PC/PE ratio. Reflecting decreased 5- and 6-desaturase activities, the 20:4(n-6)/18:2(n-6) ratio was significantly decreased by eritadenine and ethanolamine in PC of the liver microsomes, mitochondria and plasma membranes. Although the 20:4(n-6)/18:2(n-6) ratio of liver microsomal PE was also significantly decreased by eritadenine and ethanolamine, the fatty acid composition of phosphatidylinositol and phosphatidylserine was less affected by these compounds. Eritadenine and ethanolamine increased the proportion of 16:0–18:2 and decreased the proportion of 18:0–20:4 in liver PC. The results suggest that dietary eritadenine and ethanolamine might lead to decreases in desaturase activities and changes in fatty acid and molecular species composition of PC through an increase in liver microsomal PE.
KEY WORDS: • eritadenine • ethanolamine • desaturase • phosphatidylcholine • phosphatidylethanolamine • rats
Eritadenine [2(R),3(R)-dihydroxy-4-(9-adenyl)-butyric acid] is a hypocholesterolemic factor isolated from the Lentinus edodes mushroom (Shiitake in Japanese) (1
,2
), one of the most popular edible mushrooms in Japan. Eritadenine (Er)2 also exists in the Agaricus bisporus mushroom (champignon) to a lesser extent (3
). In addition to the hypocholesterolemic action, Er was found to have pronounced effects on phospholipid and fatty acid metabolism in rats as follows: 1) Er markedly depressed the synthesis of phosphatidylcholine (PC) via the phosphatidylethanolamine (PE) N-methylation pathway, thereby increasing the concentration of liver microsomal PE in vivo (4
); 2) Er decreased the PC/PE ratio of liver microsomes even when an adequate amount of choline was added to the diet (5
); and 3) Er decreased liver microsomal 6-desaturase activity with concomitant suppression of linoleic acid metabolism (6
–8
). The conversion of linoleic acid into arachidonic acid is one of the most important metabolisms of fatty acids quantitatively and qualitatively in higher animals because arachidonic acid exists in the membrane phospholipids at relatively high levels and is a major precursor for various types of eicosanoids. Hence, the suppression of linoleic acid metabolism appears to be one of the important biological effects of Er.
On the other hand, Imaizumi et al. (9
,10
) demonstrated that in addition to the hypocholesterolemic action, dietary PE increased the concentration of liver microsomal PE with a decrease in 6-desaturase activity. They also showed that ethanolamine (EA), the base constituent of PE, decreased 6-desaturase activity when added to the culture medium of rat hepatocytes (10
). Unlike Er, EA is considered to increase microsomal PE through the stimulation of PE synthesis via the cytidine diphospho (CDP)-EA pathway. Thus, previous studies led us to postulate that treatments to increase microsomal PE may necessarily decrease the activity of 6-desaturase. Although the detailed mechanism by which Er or EA decreased 6-desaturase activity remains unclear, Leikin and Brenner (11
–13
) reported that liver microsomal 5- and 6-desaturase activities may be influenced by the microsomal lipid composition, e.g., cholesterol/PC ratio or PC/PE ratio. Eritadenine supplementation decreased the PC/PE ratio of liver microsomes mainly through an increased PE concentration, rather than a decreased PC concentration, in the presence of an adequate amount of dietary choline (5
), which suggests that microsomal PE may be important in the regulation of desaturase activities. However, the relationship between the microsomal phospholipid profile and fatty acid desaturase activities has not yet been fully explored. It is noteworthy that Dobrosotskaya et al. (14
) demonstrated recently that cells of insects had a sterol regulatory element-binding protein (SREBP)-mediated regulation system that was regulated by PE rather than cholesterol and fatty acids. It is not known whether this PE-mediated regulation system exists also in mammals. Thus, it is worthwhile to verify whether the microsomal PE concentration actually participates in the regulation of lipid metabolism in mammals. PC is synthesized by either the CDP-choline pathway or the PE N-methylation pathway (15
); the latter pathway is suggested to provide specific PC molecular species (16
). Although both Er and EA decrease 6-desaturase activity, PE N-methylation is depressed only by Er. Therefore, it is interesting to know whether Er and EA differentially affect the fatty acid and molecular species profiles of PC in terms of the relative contributions of fatty acid desaturation and the PE N-methylation to PC molecular species composition.
In the present study, the effects of dietary Er and EA on liver microsomal desaturase activities and phospholipid profile were investigated comparatively in rats to test the hypothesis that an increase in microsomal PE concentration may be associated with a decrease in desaturase activities. In addition, the effects on plasma and liver lipid levels, fatty acid and molecular species composition of phospholipids were also investigated to examine whether the effect of Er was essentially the same as that of EA.
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MATERIALS AND METHODS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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[1-14C]Dihomo-
-linolenic acid, [1-14C ]linoleic acid and [1-14C]palmitic acid were obtained from New England Nuclear (Boston, MA). Unlabeled fatty acids, S-adenosylmethionine (SAM), S-adenosylhomocysteine (SAH) and EA hydrochloride were purchased from Sigma-Aldrich (St. Louis, MO). Phospholipid and fatty acid standards were purchased from Funakoshi (Tokyo, Japan). Eritadenine was kindly supplied by Tanabe Seiyaku (Osaka, Japan). All other chemicals were purchased from Wako Pure Chemical (Osaka, Japan) or Sigma-Aldrich and were of analytical grade. Mineral and vitamin mixtures (AIN-76) were purchased from oriental Yeast (Tokyo, Japan) and other ingredients of the diet were purchased from Wako or Nacalai Tesque (Kyoto, Japan).
Animals and diets.
Male 6-wk-old rats of the Wistar strain (n = 30), weighing 130–140 g, were received from Japan SLC (Hamamatsu, Japan). The rats were individually housed in hanging stainless wire cages kept in an isolated room at a controlled temperature of 23–25°C and humidity of 40–60%. Lights were maintained on a 12-h cycle (lights on from 0600 to 1800 h). For 4 or 5 d, rats were fed the powdered laboratory stock diet as described previously (17 ). Then they were divided into three groups (n = 10) with similar mean body weights (154 g) and allowed free access to the experimental diets and water for 14 d. In the present study, three experimental diets were used: a control diet, a diet supplemented with Er at a level of 0.05 g/kg and a diet supplemented with EA at a level of 8 g/kg. The composition of control diet was as follows (g/kg): casein, 250; cornstarch, 429; sucrose, 200; corn oil, 50; AIN-76 mineral mixture (18 ), 35; AIN-76 vitamin mixture (18 ), 10; choline bitartrate, 5; cellulose, 20; and lactose, 1. Eritadenine was mixed with lactose and added to the diet. Choline bitartrate was introduced into the diet at a relatively high level (5 g/kg) to avoid the development of fatty liver due to Er supplementation. The fatty acid composition of corn oil used were as follows (g/100 g fatty acids): 16:0, 12.2; 18:0, 1.8; 18:1, 32.8; 18:2(n-6), 51.4; and 18:3(n-3), 1.6. The experimental plan was approved by the Laboratory Animal Care Committee of the Faculty of Agriculture at Shizuoka University.
Tissue collection and fractionation.
At the end of the feeding period, rats that not been deprived of food were decapitated between 1200 and 1230 h. Plasma was separated from the heparinized whole blood by centrifugation at 2000 x g for 20 min at 4°C and stored at 4°C until analyses for plasma lipid concentrations. After the collection of the blood, the whole liver was quickly removed, rinsed in ice-cold saline, blotted on filter paper and weighed. A small portion of the liver (
1 g) was quickly frozen in liquid nitrogen and stored at -80°C until analyses of metabolites of methionine. The residual liver was homogenized in four volumes (vol/wt) of an ice-cold 10 mmol/L Tris-HCl buffer containing 0.25 mol/L sucrose. An aliquot (2 mL) of the homogenate was stored at -30°C until analyses for liver lipid concentrations. The subcellular fractionation was conducted essentially according to the method described by Carey and Hirschberg (19
). All centrifugations were conducted at 4°C. Another aliquot (12 mL) of the liver homogenate was filtered through four sheets of gauze to remove aggregates. The filtrate was centrifuged at 2000 x g for 10 min. An aliquot of the resulting pellet was used to purify plasma membranes; the pellet was resuspended in a small volume of the homogenizing solution, and sucrose solution (600 g/L) was added to make a final concentration of sucrose at a 470 g/L. This suspension (2 mL) was placed on the bottom of the centrifuge tube and overlaid with 410 and 82 g/L sucrose solutions (2 mL each) and centrifuged at 80,000 x g for 3 h. The band appearing at the interface of the two sucrose solutions was recovered, diluted with water to make a sucrose concentration of 0.25 mol/L and centrifuged at 2000 x g for 10 min. The resulting pellet was resuspended in the homogenizing solution. The supernatant of the first centrifugation at 2000 x g was used to obtain mitochondrial and microsomal fractions. The supernatant was centrifuged at 11,000 x g for 10 min, and the resulting pellet (mitochondrial fraction) was washed once with the homogenizing solution, recovered by the second centrifugation and resuspended in the homogenizing solution. The postmitochondrial supernatant was further centrifuged at 105,000 x g for 60 min, and the resulting pellet (microsomal fraction) was washed once with the homogenizing solution, recovered by the second centrifugation and resuspended in the homogenizing solution.
Lipid analysis.
The plasma concentrations of the total cholesterol, HDL cholesterol, triglycerides and phospholipids were measured enzymatically with kits (Cholesterol C-Test, HDL Cholesterol-Test, Triglyceride G-Test and Phospholipid B-Test, respectively; Wako Pure Chemical). The difference between total cholesterol and HDL cholesterol was assumed to be a VLDL + LDL cholesterol. The lipids of homogenates of liver and other tissues, liver microsomes, liver mitochondria and liver plasma membranes were extracted by the method of Folch et al. (20 ). The cholesterol in the extracts of the liver homogenate and microsomes was measured according to Zak (21 ). The triglycerides and phospholipids in the extract of liver homogenate were measured according to Fletcher (22 ) and Bartlett (23 ), respectively. For the determination of phospholipid class composition, the phospholipids in the extracts of liver microsomes, mitochondria and plasma membranes were separated into each class by TLC on silica gel 60 (E. Merck, Darmstadt, Germany) using chloroform/methanol/water (65:25:4, v/v/v) as a developing solvent. The bands of each phospholipid class were visualized in iodine vapor, scraped off the plate and analyzed directly for inorganic phosphorus (23 ). For the determination of fatty acid or molecular species composition, each phospholipid class was separated similarly by TLC from liver microsomes, mitochondria and plasma membranes, which were visualized with dichlorofluorescein, scraped off the plate and extracted with chloroform/methanol (1:2, v/v). For the separation of phosphatidylinositol (PI) and phosphatidylserine (PS), chloroform/acetone/methanol/acetate/water (10:4:2:2:1, v/v/v/v/v) was used as a developing solvent. The fatty acid composition of phospholipids was determined using gas-liquid chromatography as described previously (6 ). For the determination of PC molecular species composition, an aliquot of PC was converted to diacylglycerol benzoates and the derivatives were analyzed by HPLC essentially according to the method of Blank et al. (24 ) as described previously (6 ). Because some peaks consisted of two molecular species, the ratio of the two molecular species was determined by the second HPLC in which methanol/2-propanol (9:1, v/v) was used.
Assay of methionine metabolites.
The concentrations of SAM and SAH in the liver were measured with HPLC according to Cook et al. (25 ) with slight modifications; the mobile phase of HPLC was a 100 mmol/L KH2PO4 solution containing 10 mmol/L sodium heptane sulfonate and 30 mL/L methanol.
Assay of desaturase activities.
The activities of the 5-, 6-, and 9-desaturases in the liver microsomes were measured essentially according to the method of Svensson (26
) with some modifications. In brief, the reaction mixture (final 1.5 mL) contained 75 mmol/L phosphate buffer (pH 7.0), 0.15 mol/L KCl, 0.25 mol/L sucrose, 0.5 mmol/L nicotinamide, 5 mmol/L MgCl2, 1.5 mmol/L glutathione, 0.5 mmol/L CoA, 1 mmol/L NADH and microsomes (3 mg). [1-14C]Dihomo--linolenic acid, [1-14C]linoleic acid and [1-14C]palmitic acid were used as substrates for the 5-, 6-, and 9-desaturases, respectively, at a concentration of 67 µmol/L (56 MBq/mmol). After preincubation for 10 min at 37°C, the reaction was started by adding NADH, and the reaction mixture was further incubated for 20 min at 37°C in a test tube of wide bore (16 mm) under reciprocal shaking (120 cycles/min). The reaction was stopped by adding 1 mL of methanol containing KOH at a concentration of 100 g/L. After saponification and subsequent acidification, fatty acids were extracted with petroleum ether. The extracted fatty acids, together with added carrier fatty acids, were methylated with BF3/methanol reagent, and the fatty acid methyl esters were separated by HPLC as described previously (8
). Protein was measured according to Lowry et al. (27
) using bovine serum albumin as a standard.
Statistical analysis.
Data are expressed as means ± SEM. Data were analyzed by a one-way ANOVA, and the difference between means were tested using Duncan’s multiple-range test (28 ) when the F-value was significant. Simple correlation between variables was calculated by the linear regression analysis. A P-value of 0.05 or less was considered significant.
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RESULTS |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Dietary Er and EA did not affect the growth and food consumption of rats (Table 1 ). The relative liver weight was significantly lower in rats fed Er than in rats fed the control diet. Dietary Er and EA significantly decreased the plasma concentrations of the total cholesterol, VLDL + LDL cholesterol, HDL cholesterol and phospholipids, and the magnitude of the effect of Er was significantly greater than that of EA. The plasma triglyceride concentration was significantly decreased by dietary Er, but not by EA. Liver cholesterol concentration was significantly higher in rats fed Er and significantly lower in rats fed EA compared with the rats fed the control diet. The liver triglyceride concentration was significantly decreased by dietary EA, whereas the liver phospholipid concentration was significantly increased by both Er and EA.
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The concentration of SAM in the liver was significantly increased by dietary Er and conversely decreased by dietary EA (Table 1) . The concentration of SAH was markedly increased by Er and was unaffected by EA. Consequently, the SAM/SAH ratio was markedly decreased by dietary Er and slightly decreased by EA.
Phospholipid classes in the liver.
The concentration of PC in the liver microsomes, expressed in terms of microsomal protein, was significantly decreased by Er and was unaffected by EA (Table 2 ). Conversely, the concentration of PE was markedly increased by both Er and EA. The concentrations of the other phospholipids in the liver microsomes were less affected by these compounds. Neither Er nor EA affected the concentration of cholesterol in the liver microsomes. The effects of Er and EA on the phospholipid class composition in the liver microsomes, mitochondria and plasma membranes were also measured (Table 3 ). In these organelles, the proportion of PC to the total phospholipids was significantly decreased and the proportion of PE was significantly increased by both Er and EA, although the effect of Er was slightly stronger than that of EA. Consequently, the PC/PE ratio was significantly decreased in Er- or EA-fed rats.
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The proportion of 18:2(n-6) in PC was significantly increased and conversely the proportion of 20:4(n-6) in PC was significantly decreased by both Er and EA in the liver microsomes, mitochondria and plasma membranes (Table 4 ). Consequently, the 20:4(n-6)/18:2(n-6) ratio was significantly decreased in Er- or EA-fed rats. The proportion of certain longer-chain and highly unsaturated fatty acids, such as 22:5(n-6) and 22:6(n-3), in PC was also significantly decreased by Er and EA. The proportion of the saturated fatty acid 18:0 in PC was significantly decreased by both Er and EA. Conversely, the proportion of 16:0 in PC was significantly increased by Er; the proportion of 16:0 was not modified by EA except in the mitochondria. In general, the fatty acid composition of PC did not differ among the liver microsomes, mitochondria and plasma membranes, and the effect of Er was slightly stronger than that of EA. The effects of Er and EA on the fatty acid composition of PE, PI and PS in the liver microsomes were also measured (Table 5 ). Although the proportion of 18:2(n-6) in PE was markedly lower than that in PC in the control rats, the proportion of the fatty acid was significantly increased by both Er and EA. In contrast, PE contained a high level of 20:4(n-6) in control rats, and the proportion of 20:4(n-6) was significantly decreased by Er and conversely increased by EA. The proportion of 22:6(n-3) in PE was significantly increased by Er and decreased by EA. Compared with PC and PE, the proportion of polyunsaturated fatty acids (PUFA) of PI and PS was less affected by Er and EA, although saturated fatty acids were influenced by these compounds.
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The activities of the 5-, 6-, and 9-desaturases in the liver microsomes were significantly decreased by both Er and EA, and the effect of Er was greater than that of EA (Fig. 1
). There was a significant correlation between 5- or 6-desaturase activity and the proportion of PE or PC/PE ratio in the liver microsomes when the relationships between desaturase activities and microsomal lipids were analyzed using the means of the three groups (Table 7 ). Variables associated with PE generally had greater correlation coefficients than those associated with PC or cholesterol.
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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6- and 5-desaturases participate in the conversion of essential fatty acids into longer-chain and highly unsaturated fatty acids. Of these two desaturases, 6-desaturase is thought to be the rate-limiting enzyme (29
). It has been shown that 6- or 5-desaturase activity was influenced by a diabetic condition (30
) and several hormones (31
). In addition, dietary conditions or dietary supplementation with specific compounds can also affect the activity of 6- or 5-desaturase. For example, liver microsomal 6- or 5-desaturase activity was influenced by the type of dietary protein (32
) and fat (33
), choline deficiency (13
), vitamin B-6 deficiency (34
), and dietary supplementation with cholesterol (11
), phytosterols (35
), PE (10
) or Er (8
). Previously, Leikin and Brenner (11
–13
) proposed that the alteration of liver microsomal lipid composition, e.g., the cholesterol/PC or PC/PE ratio, may be associated with the changes in liver microsomal desaturase activities. The present study showed that dietary Er and EA significantly decreased all of the activities of the 5-, 6- and 9-desaturases with a concomitant alteration of the phospholipid profile, as represented by an increase in PE concentration, in liver microsomes of rats; this supports the idea that liver microsomal phospholipid composition may participate in the regulation of desaturase activities. The fact that dietary EA decreased the desaturase activities without affecting the PC concentration in liver microsomes suggests that the decrease in desaturase activities might be closely associated with the increase in PE concentration or PE-related variables, rather than PC concentration. Eritadenine increases liver microsomal PE through the suppression of PE N-methylation (4
), whereas EA is considered to increase PE through the stimulation of PE biosynthesis. Hence, it is likely that treatments to increase liver microsomal PE lead to a decrease in desaturase activities irrespective of the mechanism by which PE is increased.
It has been shown that the effect of a certain hormone or dietary cholesterol on 9-desaturase activity was different from the effect on 5- or 6-desaturase activity, although dietary PUFA are known to decrease all of the activities of the 5-, 6- and 9-desaturases. For instance, testosterone significantly increased 9-desaturase activity, whereas it significantly decreased the activities of the 5- and 6-desaturases (36
). Furthermore, dietary supplementation with cholesterol increased 9-desaturase activity and decreased the 5- and 6-desaturase activities (11
,12
). In contrast, the present study demonstrated that all of the activities of the 5-, 6- and 9-desaturases were decreased by both Er and EA, indicating that the mode of action of these compounds is different from that of testosterone or cholesterol and rather resembles that of PUFA.
It was demonstrated that PUFA decreased the activities of the 5-, 6- and 9-desaturases with a concomitant reduction of mRNA for the respective enzyme (37
–39
), indicating that the effects of PUFA are mediated by a transcriptional regulation. It was also demonstrated that the abundance of mRNA for 6-desaturase was lower in streptozotocin-induced diabetic rats than in the control rats, and this reduction of mRNA was recovered by insulin administration (40
). In contrast, Brenner et al. (41
) demonstrated recently that the suppression of 6-desaturase activity by dietary cholesterol was not accompanied by a reduction in the abundance of mRNA for the enzyme, although the stimulation of 9-desaturase activity by dietary cholesterol was followed by an increase in mRNA. In another series of experiments, we found recently that the suppression of 6-desaturase activity by dietary Er was accompanied by a significant reduction of mRNA for the enzyme (unpublished data), suggesting that unlike dietary cholesterol, a certain type of treatment to decrease 6-desaturase activity with a concomitant increase in microsomal PE concentration might elicit its effect through a transcriptional regulation. A recent report showed that SREBP, especially SREBP-1, activates the expression of both 5- and 6-desaturases in mouse liver (39
). This suggests that the suppression of desaturase activities by dietary Er or EA may also be mediated by SREBP, although it remains to be verified by further studies. Interestingly, Dobrosotskaya et al. (14
) demonstrated that cells of an insect (fruit fly, Drosophila melanogaster) had a SREBP-mediated regulation system that was regulated by PE rather than cholesterol and fatty acids. They hypothesized that the SREBP system, the transcription factor that activates genes encoding enzymes of lipid biosynthesis, may monitor the change in membrane lipids. Although it is not known whether such a PE-mediated regulation system exists also in mammals, the results obtained here may indirectly support the presence of a PE-mediated regulation system.
The present study demonstrated that the PC/PE ratio was decreased by Er and EA not only in the microsomes, but also in the mitochondria and plasma membranes in the liver in a similar manner. These results might be due to the action of phospholipid transfer proteins in the liver because phospholipid transfer proteins mediate the transfer of phospholipids, including PE, between different organelles (42
). The most prominent and common changes in PC molecular species composition caused by Er and EA were an increase in the proportion of 16:0–18:2 PC and a decrease in the proportion of 18:0–20:4 PC. The results might be due at least in part to the suppression of linoleic acid metabolism due to the decrease in 6-desaturase activity, although PC molecular species are modified by remodeling through deacylation-reacylation (43
–45
). Indeed, the effects of Er and EA on the PC molecular species profile paralleled those on the activity of 6-desaturase. In addition, it has been shown that the PC molecular species profile is also influenced by the difference in the route of PC synthesis. For instance, it was demonstrated that the CDP-choline pathway produced more 16:0–18:2 and 16:0–18:1 PC molecular species and, conversely, the PE N-methylation pathway produced more 18:0–20:4 and 18:0–22:6 PC molecular species than the alternative pathways in the primary cultured rat hepatocytes (16
). Previously, we demonstrated that Er depressed the synthesis of PC via the PE N-methylation pathway by >90% in vivo (4
), indicating that PC synthesis is largely dependent on the CDP-choline pathway in Er-fed rats. Ethanolamine is thought to stimulate PE synthesis via the CDP-EA pathway, but EA could not stimulate the conversion of PE into PC in isolated rat hepatocytes (46
). The results that changes in liver PC molecular species composition caused by Er and EA were similar suggest that EA does not stimulate the production of PE N-methylation pathway-associated specific PC molecular species, which supports that EA may not stimulate the conversion of PE into PC even in vivo. Thus, it is likely that EA altered the PC molecular species profile mainly through the depression of linoleic acid metabolism, and that this might also be the case for Er. However, the effect of cessation of PE N-methylation may participate in the effect of Er to some extent because Er increased the proportion of 22:6(n-3) in liver microsomal PE and conversely decreased the proportion of 22:6(n-3) in liver microsomal PC (Tables 5
and 6)
.
The detailed mechanisms by which Er and EA elicit their hypocholesterolemic action have not yet been fully elucidated. However, the hypocholesterolemic action of Er might be elicited through an enhanced uptake of plasma lipoprotein cholesterol by tissues, especially by the liver because cholesterogenesis in the liver (47 ), excretion of steroids into feces (48 ) and secretion of lipoprotein cholesterol from the liver (unpublished data) were not affected by Er. This deduced mechanism might also be applicable to the hypocholesterolemic action of EA because dietary PE compared with PC did not increase fecal steroid excretion (9 ), and soybean phospholipids containing PE compared with soybean oil did not depress the secretion of triglyceride-rich lipoprotein from the liver as measured by the injection of Triton WR-1339 (49 ). It is of interest whether the increase in microsomal PE concentration and the suppression of linoleic acid metabolism, the common effects of Er and EA, are associated with the hypocholesterolemic action of these compounds. Indeed, other dietary treatments that increase microsomal PE, e.g., soybean protein (17 ,50 ) and glycine (51 ), also decrease plasma cholesterol concentration. Soybean protein induces a low hepatic SAM concentration (17 ,50 ) and glycine competes with PE for SAM (52 ), thereby reducing the PE N-methylation reaction. These results, together with those on the effects of Er and EA, strongly suggest that treatments that increase liver microsomal PE concentration might necessarily reduce plasma cholesterol. However, it is difficult to distinguish the effect of increased microsomal PE concentration from that of suppressed linoleic acid metabolism because the former was accompanied by a suppression of linoleic acid metabolism as discussed above.
At least two possible mechanisms exist for the increased uptake of plasma lipoprotein cholesterol by the liver, i.e., the increased activity of lipoprotein receptors and the increased uptake of plasma lipoprotein cholesterol due to changes in the nature of plasma lipoproteins. Currently, it is not known whether the increased microsomal PE concentration per se is associated with the hypocholesterolemic action of Er or EA. On the other hand, several reports have shown that the uptake rate of plasma lipoprotein lipids was influenced by the molecular species of plasma lipoprotein PC. For instance, the uptake rate of cholesteryl oleate of reconstituted HDL by perfused rat livers was most stimulated by 16:0–18:2 PC of several molecular species tested to reconstitute HDL particles (53 ). These results were due to the role of hepatic lipase in the uptake of HDL cholesterol by the liver because hepatic lipase has a phospholipase A1 activity, in addition to triglyceride lipase activity, and the hydrolysis of PC of HDL stimulates the uptake of HDL lipids by the liver. In fact, 16:0–18:2 PC was hydrolyzed by hepatic lipase at the highest rate of several PC molecular species tested (53 ). Thus, the 16:0–18:2 PC appears to be the key molecular species in stimulating the metabolism of HDL cholesterol. Although the molecular species composition of plasma PC was not determined in the present study, the proportion of 16:0–18:2 molecular species in plasma PC increased markedly in Er-fed rats (6 ). Therefore, the possibility that Er and EA may increase the uptake of plasma HDL cholesterol through the alteration of the PC molecular species profile of HDL cannot be excluded. In contrast, the hypolipidemic action of dietary fish oil, which contains eicosapentaenoic acid and docosahexaenoic acid, likely resulted from the suppressed expression of cholesterogenic and lipogenic enzymes through a decrease in SREBP in mice (54 ). It remains to be investigated whether the hypocholesterolemic action of Er, EA or other treatments to increase liver microsomal PE is mediated by a similar transcriptional regulation.
In conclusion, the results obtained here indicate that dietary Er and EA cause decreases in desaturase activities and changes in fatty acid and molecular species profiles of PC through an increase in liver microsomal PE concentration in rats. The results also suggest that in addition to its activity in insects (14 ), microsomal PE concentration may be one of the factors that affect the metabolism of lipids in mammals.
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FOOTNOTES |
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Manuscript received 19 September 2002. Initial review completed 31 October 2002. Revision accepted 2 December 2002.
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LITERATURE CITED |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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