The Journal of Nutrition Vol. 129 No. 1 January 1999, pp. 146-151

Hepatic Fatty Acid Oxidation Enzyme Activities Are Stimulated in Rats Fed the Brown Seaweed, Undaria pinnatifida (Wakame)^1,2

Masakazu Murata³, Kenji Ishihara, and Hiroaki Saito

Laboratory of Lipid Chemistry, Marine Biochemistry Division, National Research Institute of Fisheries Science, Yokohama 236-8648, Japan

	ABSTRACT

Abstract Introduction Methods Results Discussion References

The activities of hepatic enzymes involved in fatty acid synthesis and oxidation were compared in rats fed diets containing different proportions of dried powder of the brown seaweed, Undaria pinnatifida (wakame). Rats were fed diets containing 0, 0.5, 1.0, 2.0, 5.0 and 10 g/100 g of dried wakame powder. Experimental diets were adjusted to provide consistent amounts of most nutrients, but mineral concentrations were not standardized. After the 21-d feeding period, serum and liver triacylglycerol levels in rats fed diets in which wakame constituted at least 2% were significantly lower than those in rats fed the control diet. The activity of glucose-6-phosphate dehydrogenase was significantly lower in rats fed the 5 and 10% wakame diets than in rats fed the control diet. In contrast, 10% wakame diet increased activities of enzymes involved in the -oxidation pathway including hepatic carnitine palmitoyltransferase, acyl-CoA dehydrogenase, acyl-CoA oxidase, enoyl-CoA hydratase and 2,4-dienoyl-CoA reductase. Some differences were detected in rats fed 5% wakame as well. These results suggest that alterations of the activities of enzymes involved in fatty acid metabolism in the liver are responsible for the serum triacylglycerol-lowering effect of dietary wakame. Thus, wakame may be useful as a food to prevent hyperlipidemia.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

The Japanese customarily eat seaweed, and the number of species eaten has been estimated at more than 20. In particular, Undaria pinnatifida, (wakame) is one of the most widely eaten edible brown seaweeds in Japan. The part of wakame used for food is the sterile leaf of the asexual generation. Wakame is marketed in a dried or salted state for long preservation. Dried or salted wakame is softened by flushing the salt with water and is eaten in seafood salad with vegetables or as an ingredient of miso soup in Japan.

In the past 20 yr, the intake of seaweeds decreased as the intake of dairy products and meat increased in Japan. Along with the change in eating habits, the incidence rates of diseases such as hyperlipidemia, arterialsclerosis and coronary arterial disease are increasing. In particular, hyperlipidemia, which is a preliminary symptom of arterialsclerosis and cardiac disease, is increasing remarkably. Therefore, a search for effective food materials for the treatment of hyperlipidemia is necessary.

Recently, elements which maintain homeostasis in organisms were reported to be included in wakame. Elements in wakame were found to have coagulation protective action for human blood (Hori and Nishizawa 1982), antitumor action (Usui et al. 1980) and antimutagenic activity (Okai et al. 1993). In addition, (n-3) polyunsaturated fatty acids, which have antithrombus, antihypercholesterolemia and antiallergy action, are present in wakame. Moreover, wakame includes dietary fiber assumed to suppress the absorption of cholesterol from the intestinal wall. However, few reports exist concerning the influence of wakame on the metabolism of lipids other than cholesterol, although wakame is believed to contain elements which regulate lipid metabolism.

In this study, we examined the influence of wakame on lipid metabolism to clarify if it could be used as a food to prevent hyperlipidemia.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Materials.  The wakame powder was a gift from the Research Conference for Nutrition and Health of Wakame (Tokyo, Japan). The constituents (g/100 g) of wakame were 17.2 g of protein, 3.7 g of lipid, 40.6 g of carbohydrate, 3.1 g of fiber and 5.0 g of minerals (Standard tables of food composition in Japan 1982). Palmitoyl-CoA (16:0-CoA)⁴, arachidoyl-CoA (20:4-CoA), eicosapentaoyl-CoA (EPA-CoA) and docosahexaoyl-CoA (DHA-CoA) were prepared according to the method of Kawaguchi et al. (1981). Acetyl-CoA was prepared by acylating CoA with acetic anhydride, acetoacetyl-CoA was prepared using diketene and crotonyl-CoA using crotonic anhydride. Sorboyl-CoA was prepared using the mixed anhydride method (Goldman and Vagelos 1961). Malonyl-CoA was purchased from the Sigma Chemical Co. (St. Louis, MO). Bovine serum albumin fraction V (essential fatty acid-free) was the product of Boehringer Mannheim (GmbH, Germany).

Animals and diets.  Male Sprague-Dawley rats obtained from the Charles River (Kanagawa, Japan) were housed individually in stainless-steel mesh cages in an air-conditioned room (temperature: 20-22°C; humidity: 55-65%; lighting: 0700-1900) and were fed on a commercial nonpurified diet (Type NMF; Oriental Yeast Co., Tokyo, Japan). After 7 d of acclimation to the housing conditions, rats were fed purified experimental diets. Six groups of 7 rats were fed purified experimental diets containing wakame powder. Nutrient contents were standardized by subtracting the amount of nutrients included in the wakame from the basic diet. However, mineral concentration likely differed among experimental diets since wakame contains various minerals. In the present study, we did not adjust the mineral contents, since it was impossible to completely standardize mineral contents among all experimental diets.

The composition of the basic experimental diet was (g/100 g diet): vitamin-free casein, 20; rapeseed oil, 5; AIN-76 mineral mixture, 3.5; AIN-76 vitamin mixture, 1; cellulose powder, 5; gelatinized potato starch, 15; DL-methionine, 0.3; choline bitartrate, 0.2; and sucrose, 50. The compositions of the mineral and vitamin mixtures were those recommended by the American Institute of Nutrition (1977). Each dietary composition group is shown in Table 1. All the diet ingredients were products of the Oriental Yeast Co. (Tokyo, Japan). The average body weight of rats at the beginning of experiments was 153 ± 11 g. The care and treatment of experimental animals conformed to the National Research Institute of Fisheries Science (Yokohama, Japan) guidelines for the ethical treatment of laboratory animals.

Enzyme assay.  At the end of the experimental period, rats were lightly anesthetized with diethylether, bled from the abdominal aorta and livers were quickly excised. About 3 g of liver from rats fed 0, 5.0 or 10.0% wakame-containing diet was homogenized with 7 vol of 0.25 mol/L sucrose and centrifuged at 500 × g for 10 min. The supernatant was recentifuged at 9000 × g for 10 min to isolate mitochondria. The mitochondrial fraction was washed twice with 0.25 mol/L sucrose containing 1 mmol/L EDTA and 3 mmol/L Tris-HCl (pH 7.0) and finally suspended in the same medium to give a protein concentration of 20-25 g/L. Glucose 6-phosphate dehydrogenase (EC 1.1.1.49) (Kelley and Kletzien 1984), malic enzyme (EC 1.1.40) (Hsu and Lardy 1969) and fatty acid synthetase (Kelly et al. 1986) activities were measured in the 9000 × g supernatant fraction of liver homogenates (Ide et al. 1992).

The supernatant fraction obtained after centrifugation of liver homogenates at 500 × g for 10 min was used for measurement of the activities of fatty acid oxidation enzymes except for carnitine palmitoyltransferase (EC 2.3.1.21) and acyl-CoA dehydrogenase (EC 1.3.99.3). Because carnitine palmitoyltransferase and acyl-CoA dehydrogenase are primarily mitochondrial enzymes (Schulz 1991) and assays using the 500 × g supernatant fraction as the enzyme source gave extremely high blank values, the above two enzymes were assayed using the isolated mitochondrial fraction as an enzyme source. Carnitine palmitoyltransferase activity was measured in the isolated mitochondrial fraction solubilized with Triton X-100 according to methods as described elsewhere (Ide et al. 1987). This assay may detect not only transferase I activity but also that of the enzyme located in the inner mitochondrial membrane (carnitine palmitoyltransferase II). Acyl-CoA dehydrogenase (EC 1.3.99.3) activity was measured in isolated mitochondrial fraction according to the method described by Dommes and Kumnau (1976) and Dommes et al. (1981) except that phenazine methosulfate was used as a primary electron acceptor. Acyl-CoA oxidase (EC 1.3.3.6) activity was measured in the 500 × g supernatant fraction of liver homogenates as described elsewhere (Hashimoto et al. 1981, Ide et al. 1987). 16:0-CoA was used as a substrate for the carnitine palmitoyltransferase assay, and 16:0-CoA, 20:4-CoA, EPA-CoA and DHA-CoA were used as substrates for the acyl-CoA dehydrogenase and acyl-CoA oxidase assays. Crotonyl-CoA was used as a substrate for enoyl-CoA hydratase (EC 4.2.1.17) (Osumi and Hashimoto 1979a), acetoacetyl-CoA for 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) (Osumi and Hashimoto 1979), and sorboyl-CoA for 2,4-dienoyl-CoA reductase (EC 1.3.1.34) (Kunau and Dommes 1978) in assaying the activities. Activities of marker enzymes for cell organelles including succinate dehydrogenase (EC 1.3.99.1) (mitochondria) (Vegger et al. 1969) and catalase (EC 1.11.1.6) (peroxisomes) (Cohen et al. 1970) were determined in the 500 × g supernatant fraction of liver homogenates.

Lipid analyses.  Liver and serum lipids were extracted and purified (Folch et al. 1957). Triacylglycerol, phospholipid and cholesterol contents in the extracts were determined as described previously (Hara et al. 1993). Fatty acid composition of serum and liver lipids was determined using gas-liquid chromatography (Murata and Ide 1997). The -hydroxybutyrate in serum was measured enzymatically in a deproteinized sample as described elsewhere (Ide and Ontko 1981).

Statistical analyses.  All values are expressed as mean ± (SEM). Differences among dietary groups (P < 0.05) were analyzed by analysis of variance and Duncan's multiple range test (Steel and Torrie 1980). The analyses were performed by the statistics programs of Ide et al. (1995) using Microsoft Excel (Microsoft Corp., Redmond, WA).

	RESULTS

Abstract Introduction Methods Results Discussion References

Rats were fed diets containing 0, 0.5, 1.0, 2.0, 5.0 and 10% wakame for 21 d. Dietary wakame did not affect body weight gains (273 ± 7 g, n = 42) or the amount of experimental diets consumed during the 21 d (531 ± 16 g, n = 42). The weights of livers excised at the end of the experimental period did not differ in rats fed diets containing different proportions of wakame powder and control diet (data not shown).

Serum and liver triacylglycerol levels decreased significantly as the dietary levels of wakame increased (Table 2). The addition to the diet of wakame at levels of at least 2% significantly decreased rat serum triacylglycerol compared with the control diet. The addition of wakame powder to the diet at levels of at least 1.0% significantly decreased the concentrations of rat liver triacylglycerol compared with the control diet. In contrast, there were no significant differences in the concentration of cholesterol and phospholipid in serum or liver from rats fed wakame and control diets except for liver cholesterol in rats fed 10% wakame diet which was lower than in controls.

The concentrations of -hydroxybutyrate in serum of rats fed diets with 5.0 and 10% wakame were higher than that of rats fed control diet (Table 3). Dietary wakame did not affect the activities of hepatic malic enzyme or fatty acid synthetase, but the activity of glucose-6-phosphate dehydrogenase, which provides NADH for fatty acid synthesis, was significantly lower in rats fed 5 and 10% wakame diets compared with that in rats fed the control diet (Table 3).

View this table: [in this window] [in a new window]   Table 3. Concentration of -hydroxybutyrate in serum and activities of enzymes involved in fatty acid synthesis in the liver of rats fed diets containing different proportions of wakame1

The activities of various enzymes involved in the fatty acid -oxidation pathway in rats fed diets containing 0, 5.0 and 10.0% wakame are shown in Table 4. Protein contents in mitochondrial fraction in rats fed the wakame diets were not different from that in rats fed the control diet [5% wakame diet; 19.2 ± 1.5, 10% wakame diet; 20.5 ± 2.4, control diet; 19.8 ± 2.4 (mg/g liver)].

The activity of carnitine palmitoyltransferase, which regulates the rate of transport of fatty acids across the mitochondrial membrane, was significantly greater in rats fed 10.0% wakame diet compared with those fed the control diet (Table 4). The 10 and 5% wakame diets also increased the activity of acyl-CoA dehydrogenase, which was the main enzyme involved in fatty acid oxidation in mitochondria when all fatty acid CoA were used as substrates. However, the specific activities of acyl-CoA dehydrogenase were in the order 16:0-CoA > EPA-CoA > 20:4-CoA > DHA-CoA as was the extent of increased activities of acyl-CoA dehydrogenase in rats fed the 10% wakame diet.

The same tendency was observed in the activity of Acyl-CoA oxidase, which is the rate-limiting enzyme for fatty acid -oxidation in peroxisomes. Rats fed 10% wakame had significantly (P < 0.05) greater acyl-CoA oxidase activity when all fatty acid CoA were used as substrates, and those fed 5% wakame had greater activity when two of the four substrates were used (Table 4). The specific activities of this enzyme were in the order 16:0-CoA > 20:4-CoA = DHA-CoA > EPA-CoA, and the extent of increased activities were in the same order in rats fed the 10% wakame diet.

The activity of 2,4-dienoyl-CoA reductase, which degrades unsaturated fatty acids having cis double bond(s) via the -oxidation pathway, was significantly (P < 0.05) greater in rats fed 5 and 10% wakame diets compared with those fed the control diet. The 5 and 10% wakame diets also significantly (P < 0.05) increased the activity of enoyl-CoA hydratase.

The specific activities of succinate dehydrogenase [(control diet; 68.0 ± 6.2, 5% wakame diet; 65.7 ± 5.7, 10% wakame diet; 70.2 ± 6.6 (µmol/(min·mg protein)] and catalase [(control diet; 0.41 ± 0.02, 5% wakame diet; 0.45 ± 0.03, 10% wakame diet; 0.48 ± 0.04 (Kat/mg protein)] did not differ among experimental groups.

The 10% wakame diet increased the proportion of arachidonic acid in serum lipids (Table 5). The 5 and 10% wakame diets increased the proportions of stearic acid, linoleic acid and arachidonic acid with an accompanying decrease in proportions of palmitic and oleic acids in liver lipids (Table 5). The proportion of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in liver lipids from rats fed diet containing wakame was slightly but significantly (P < 0.05) higher than those in rats fed the control diet.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

Addition of 2% wakame to rat diets significantly decreased serum and liver triacylglycerol concentrations. Furthermore, the extent of the decreases in serum and liver triacylglycerol concentrations increased as the amount of wakame added to the diet increased. However, dietary wakame did not affect the food intake or body weight gain of rats. Therefore, it is unlikely that a difference in energy consumption is a factor in the serum and liver triacylglycerol-lowering effects of dietary wakame.

In contrast, dietary wakame did not influence the concentration of serum cholesterol in the present study. We thought that rat serum cholesterol concentration would be decreased by dietary wakame, since fucosterol (Tsuda et al. 1957) and/or dietary fiber are part of wakame and both decrease the concentration of serum cholesterol by decreasing the absorption of cholesterol from the small intestine. Therefore, if cholesterol is added to the experimental diet, dietary wakame may decrease concentration of serum cholesterol.

The absorption of lipid from the small intestine and/or metabolism of lipids and fatty acids in the liver affect serum and liver triacylglycerol concentrations. Therefore, it is possible that dietary wakame modifies the rates of synthesis and degradation of fatty acid and lipids in the liver. Evidence supports the idea that the rates of fatty acid synthesis and oxidation are modified under the above conditions. Starvation (Ide et al. 1980) and experimental diabetes (Nepokroeff et al. 1974) reduce the rate of fatty acid synthesis but increase the rate of fatty acid oxidation in the liver (Mayers and Felrs 1967, Van Harken et al. 1969). Dietary fish oil decreases the rate of ³H₂O incorporation into fatty acids but increases ketone body production in perfused rat hepatocytes (Wong et al. 1984). Numerous studies also indicate that fish oil increases the rate of fatty acid oxidation in rat liver peroxisomes (Halminski et al. 1991, Rustan et al. 1992). These nutritional and pathological conditions accompany the reduction in the rate of very low density lipoprotein (VLDL) production in the liver (Mayers and Felrs 1967, Van Harken et al. 1969, Wong et al. 1984). Conversely, feeding (Ide et al. 1980) and treatment of diabetes by insulin (Nepokroeff et al. 1974) enhance the rate of fatty acid synthesis but decrease the rate of fatty acid oxidation accompanying the increase in the rate of VLDL production in the liver (Mayers and Felrs 1967, Van Harken et al. 1969). In the present study, reciprocal responses in enzymes of fatty acid synthesis and oxidation in the liver were observed in rats fed wakame diets and the control diet. Therefore probably dietary wakame modifies fatty acid synthesis and oxidation in the liver, and these changes contribute to the serum and liver triacyglycerol-lowering effects.

In this study, we examine the effect of dietary wakame on the activities of enzymes involved in fatty acid synthesis. Dietary wakame significantly decreased the activity of glucose-6-phosphate dehydrogenase compared with the control diet. However, no differences existed in the activities of either malic enzyme or fatty acid synthetase. Therefore, the change in the fatty acid synthesis was not a major factor responsible for the serum and liver triacylglycerol-lowering effects. In contrast, dietary wakame increased the concentration of -hydroxybutylate in serum, indicating that fatty acid -oxidation in the liver was augmented by dietary wakame.

Next, we examined the influence of dietary wakame on the activities of enzymes involved in fatty acid -oxidation in rat livers: an increase in various enzymes' activities. In particular, a remarkable increase occurred in the activity of acyl-CoA dehydrogenase the main enzyme involved in fatty acid -oxidation in mitochondria in rats fed 10% wakame diet. Furthermore, the extent of increased activity of acyl-CoA dehydrogenase using 16:0-CoA as a substrate was one to two times higher than those using unsaturated fatty acid CoA, 20:4-CoA, EPA-CoA and DHA-CoA, as substrates. The specificity of acyl-CoA dehydrogenase to the 16:0-CoA than unsaturated fatty acid-CoA observed was consisted with data reported previously (Izai et al. 1992). Dietary wakame also seemed to increase the specificity of acyl-CoA dehydrogenase for the 16:0-CoA. However, acyl-CoA oxidase activities in rats fed wakame diet increased by only 30-50% greater than those in rats fed the control diet, and the proportionate increase in activity of acyl-CoA oxidase by dietary wakame was lower than that of acyl-CoA dehydrogenase. Therefore, we propose that dietary wakame dramatically increased -oxidation of saturated fatty acids in the mitochondria.

Dietary wakame did not increase the activities of marker enzymes for mitochondria (succinate dehydrogenase) and peroxisomes (catalase), so it may specifically induce enzymes in the -oxidation pathway without causing the proliferation of these cell organelles.

These results were reflected in the serum and liver lipid fatty acid compositions. The wakame diets significantly (P < 0.05) modified the fatty acid compositions of serum and liver lipids in spite of the fact that the fatty acid compositions of these diets were comparable. 2,4-Dienoyl-CoA reductase is a rate-limiting enzyme for the oxidation of unsaturated fatty acids and exists in both the mitochondrial matrix and peroxisomes. Dietary wakame significantly (P < 0.05) increased the activity of this enzyme compared with the control diet. However, the proportionate increase in the activity of this enzyme was smaller than that of acyl-CoA dehydrogenase assayed using 16:0-CoA as substrate. Therefore, we suggest that dietary wakame caused palmitic acid to be oxidized by the liver more promptly than unsaturated fatty acids. These changes in the oxidation of fatty acids in the liver may account for the decrease in the proportion of palmitic acid to total fatty acids in rats fed wakame diets. Consequently, dietary wakame may decrease the proportion of palmitic acid in liver lipids. The tendency of dietary wakame to reduce the rate of fatty acid synthesis, as expected from the reduction in glucose-6-phosphate dehydrogenase, may increase the relative availability of linoleic acid as a substrate for enzymes involved in glycerolipid biosynthesis and fatty acid desaturation and elongation. Consequently the proportion of linoleic acid and arachidonic acid in rats fed dietary wakame may increase.

We also observed a decrease in the proportion of oleic acid to total fatty acids in serum and liver lipids in rats fed wakame diets. Therefore, examination of the specificity of oleic acid for enzymes involved in fatty acid -oxidation in rats fed wakame diet is necessary.

Malonyl-CoA regulates the mitochondrial oxidation of long-chain fatty acids by inhibiting the activity of carnitine palmitoyltransferase (Schultz 1991). The concentration of malonyl-CoA in the liver, which is the enzyme product of acetyl-CoA carboxylase, decreases proportionally as the rate of fatty acid synthesis decreases (Malewiak et al. 1985, Schulz 1991). In the present study, dietary wakame decreased the activity of glucose-6-phosphate dehydrogenase involved in fatty acid synthesis compared to the control, indicating that dietary wakame decreased fatty acid synthesis in the liver. Although we did not measure the activity of acetyl-CoA carboxylase, probably dietary wakame also decreases the activity of this enzyme in the liver. Thus, we suggest that dietary wakame mediated the inhibition of fatty acid synthesis which accompanied the reduction in malonyl-CoA concentration in the liver. Measurement of acetyl-CoA carboxylase activity and concentration of malonyl-CoA in the liver in rats fed wakame are required to clarify this point.

Reportedly, dietary fish oil, which contains polyunsaturated fatty acids, markedly decreased rat serum and liver triacylglycerol concentrations, and the decrease was caused by accelerated polyunsaturated fatty acid oxidation in liver peroxisomes (Halminski et al. 1991, Rustan et al. 1992). The serum and liver triacylglycerol-lowering effects were also confirmed by providing wakame in the present experiment; however, these effects could be dissociated from induction of mitochondrial fatty acid -oxidation.

The wakame powder contains a considerable amount of unsaturated fatty acid, 18:2(n-6), 6.7%; 18:3(n-3), 12.4%; 20:4(n-6), 9.9%; and EPA, 10.6% of the total fatty acids, according to our analysis. However, the proportions of 18:3(n-3) and EPA from wakame powder, which alter rat lipid metabolism, are 0.87 and 0.74% of the total fatty acids in the 10% wakame diet, respectively. Therefore, it is improbable that these fatty acids from the wakame powder alter rat lipid metabolism.

The mineral contents of each experimental diet were not standardized in the present study, with the 10% wakame diet containing a large amount of minerals. Namely, the concentration (mg/100 g diet) of calcium was 137.0 and 18.2, phosphorus was 46.0 and 14.0, sodium was 277.0 and 3.6, and potassium was 142.0 and 12.6 in the 10% wakame and control diets, respectively. Therefore, examination of the influence of the minerals in wakame powder on the metabolism of rat lipid is necessary to clarify the mechanism of the serum and liver triacylglycerol-lowering effects of dietary wakame.

In summary, dietary wakame decreased rat serum and liver triacylglycerol levels. Further, dietary wakame increased the activities of enzymes involved in the fatty acid oxidation in mitochondria. Thus, the increased fatty acid oxidation in liver mitochondria is a factor responsible for the serum and liver triacylglycerol-lowering effects of dietary wakame. We believe it necessary to examine, in detail the influence of various elements in wakame on lipid metabolism.

	FOOTNOTES

Manuscript received 19 May 1998. Initial reviews completed 8 July 1998. Revision accepted 14 October 1998.

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