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© 2004 The American Society for Nutritional Sciences J. Nutr. 134:1320-1327, June 2004

Biochemical and Molecular Actions of Nutrients

Fish Protein Hydrolysate Reduces Plasma Total Cholesterol, Increases the Proportion of HDL Cholesterol, and Lowers Acyl-CoA:Cholesterol Acyltransferase Activity in Liver of Zucker Rats1

Hege Wergedahl2, Bjørn Liaset*,3, Oddrun Anita Gudbrandsen3, Einar Lied{dagger}, Marit Espe*, Ziad Muna, Sverre Mørk** and Rolf K. Berge

Institute of Medicine, Section of Medical Biochemistry, University of Bergen, Haukeland University Hospital, Norway; * National Institute of Nutrition and Seafood Research, Bergen, Norway; {dagger} NutriMarine Life Science, Bergen, Norway; and ** Gade Institute, Department of Pathology, University of Bergen, Haukeland University Hospital, Norway

2To whom correspondence should be addressed. E-mail: hege.vagenes{at}ikb.uib.no.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
There is growing evidence that soy protein improves the blood lipid profiles of animals and humans. We compared the effects of fish protein hydrolysate (FPH), soy protein, and casein (control) on lipid metabolism in Wistar rats and genetically obese Zucker (fa/fa) rats. In Zucker rats, FPH treatment affected the fatty acid composition in liver, plasma, and triacylglycerol-rich lipoproteins. The mRNA levels of {Delta}5 and {Delta}6 desaturases were reduced by FPH and soy protein feeding compared with casein feeding. In Zucker rats both FPH and soy protein treatment reduced the plasma cholesterol level. Furthermore, the HDL cholesterol:total cholesterol ratio was greater in these rats and in the Wistar rats fed FPH and soy protein compared with those fed casein. Although fecal total bile acids were greater in soy protein–fed Zucker rats than in casein-fed controls, those fed FPH did not differ from the controls. However, the acyl-CoA:cholesterol acyltransferase activity was reduced in Zucker rats fed FPH and tended to be lower (P = 0.13) in those fed soy protein compared with those fed casein. Low ratios of methionine to glycine and lysine to arginine in the FPH and soy protein diets, compared with the casein diet, may be involved in lowering the plasma cholesterol concentration. Our results indicate that the effects of FPH and soy protein on fatty acid metabolism are similar in many respects, but the hypocholesterolemic effects of FPH and soy protein appear to be due to different mechanisms. FPH may have a role as a cardioprotective nutrient.

KEY WORDS: • plasma cholesterol • fish protein • fatty acid composition • fecal bile acids

The hypocholesterolemic effect of soy protein has been demonstrated in numerous studies in animal models (1) and humans (2). Relative to casein, dietary soy protein reduces plasma cholesterol, but increases cholesterol synthesis, induces LDL receptor expression, increases bile acid synthesis, and decreases steroid absorption from the intestine (3). These events may together reduce the plasma total cholesterol in rats. The specific soy constituents responsible for the changes in blood lipids remain to be identified. Several studies suggest, however, that amino acids or peptides may be the bioactive components of soy protein. Dietary substitution of amino acid mixtures mimicking soy protein significantly lowered the cholesterol concentrations in blood compared with amino acid mixtures simulating casein. However, the resulting cholesterol concentrations in rats or rabbits fed the soy amino acids were not as low as those in animals fed the intact protein (4,5). Furthermore, when an amino acid mixture corresponding to soy protein was fed to rats, cholesterol absorption and fecal excretion were not affected (4). However, feeding rats the indigestible fractions of soy protein increased the fecal excretion of sterols, and lowered blood and liver cholesterol concentrations (6). In humans, the cholesterol-lowering effect of soy protein occurred primarily in persons who were hypercholesterolemic before dietary intervention (2). Hyperlipidemia is associated with the development of atherosclerosis, cardiovascular disease (CVD),4 and noninsulin-dependent diabetes mellitus (NIDDM) (7), and a hypocholesterolemic effect of soy protein may therefore lower the risk of CVD and NIDDM. Obese Zucker rats are hyperlipidemic (8) and develop hepatic steatosis within a few weeks of birth (9). Mechanisms responsible for the effects of soy protein on CVD and NIDDM risk involve beneficial changes in liver and plasma lipid metabolism. We therefore used Zucker rats as a model system (10).

Although the cholesterol-lowering effect of soy protein compared with casein is well known, only a few studies have reported a hypolipidemic effect of fish protein (11,12). We investigated the hypocholesterolemic effects of a fat-free hydrolyzed muscle protein obtained from Atlantic salmon (FPH). First, a pilot experiment was performed in Wistar rats, testing the hypolipidemic effect of FPH vs. soy protein and casein. Obese Zucker rats were used to further investigate the effect of FPH and soy protein on lipid metabolism in a hyperlipidemic model. The effect of FPH on plasma cholesterol and the activities of enzymes involved in cholesterol metabolism were investigated. Total fecal bile acids were measured in Zucker rats because the major route of elimination of cholesterol from the body is fecal excretion of cholesterol as bile acids. It was of particular interest to examine whether the hypocholesterolemic effects of FPH and soy protein operate by the same mechanism. Fatty acid oxidation is important for the lipid-lowering effect of hypolipidemic drugs in rats (13), whereas impaired fatty acid oxidation causes fatty liver and an increased level of plasma triacylglycerol in rats (14). Because lipogenesis and fatty acid esterification are enhanced in the liver of obese Zucker rats (15) and fatty acid oxidation is depressed (16), we measured the activity of enzymes involved in ß-oxidation and lipogenesis in Zucker rats fed the different dietary proteins. Additionally, the fatty acid composition of liver, plasma, and triacylglycerol-rich lipoproteins was established to determine whether FPH affected the fatty acid profile.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
    Chemicals. Fish protein hydrolysate (FPH) was produced from fish flesh remnants on salmon bone frames after filleting, as described previously (17,18); the Supro 530 EX soy protein was from DuPont Protein Technologies; the Danpro A soy concentrate was from Central Soy European Proteins; and bovine casein sodium salt, C-8654, was from Sigma-Aldrich. The chemicals used for real-time RT-PCR were from Applied Biosystems. Radiolabeled substrates were purchased from Amersham. All other chemicals were obtained from common commercial sources and were of reagent grade.

    Animals and treatments. Male lean Wistar rats from Möllegaard breeding Laboratory, weighing 85 ± 5 g, and male obese fa/fa Zucker rats from Charles River, weighing 120 ± 3 g, were kept in a room maintained on a 12-h light:dark cycle, at a temperature of 20 ± 3°C, and relative humidity of 65 ± 15%. The day after arrival, the rats were placed in separate metabolic cages; then they were divided into experimental groups (n = 6/group). The rats were adapted to the experimental conditions and diets for 4 d. The semipurified diets (Table 1) contained 20 g/100 g crude protein (N x 6.25) in the form of FPH, soy, or casein. The partial amino acid composition of the different diets is given in Table 2. The rats were offered equal feed rations daily, and the amount fed was adjusted to meet the expected consumption based on the feed intake on the previous day. The rats had free access to water. The Wistar rats were fed for 11–12 d, and the Zucker rats were fed for 22 or 23 d after acclimation (3 rats from each group were killed on d 1 and the rest on d 2). At the end of the feeding period, the rats were anesthetized subcutaneously by 1:1 Hypnorm/Dormicum (Fentanyl/fluanisone-Midazolam), 2 mL/kg body weight. Cardiac puncture was performed to collect blood samples in a syringe containing heparin, and the plasma samples were stored at –80°C until analysis. The liver was dissected, and a portion was immediately frozen in liquid nitrogen; the rest of the liver was chilled on ice for homogenization. The protocol was approved by the Norwegian State Board of Biological Experiments with Living Animals.


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  		TABLE 1 Composition of the experimental diets1

 

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  		TABLE 2 Amino acid composition of the experimental diets1

 
    Preparation of subcellular fractions. Homogenization and subcellular fractionation of the livers was performed as previously described (19). The procedure was performed at 0–4°C, and the fractions were stored at –80°C. Protein was assayed with the BioRad protein assay kit using bovine serum albumin as the standard.

    Enzyme assays. Carnitine palmitoyltransferase (CPT)-I and CPT-II activities were measured in the postnuclear (Wistar rats) or mitochondrial (Zucker rats) fraction (20). Acyl-CoA:cholesterol acyltransferase (ACAT) was measured in the microsomal fraction (21), 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) was measured in the microsomal fraction (22), and HMG-CoA synthase was measured in the mitochondrial fraction (23). Fatty acid synthase was measured in the cytosolic fraction as described by Roncari (24), modified according to Skorve et al. (25), and acetyl-CoA carboxylase was determined in the cytosolic and mitochondrial fractions by measuring the amount of NaH14CO3 incorporated into malonyl-CoA (26).

    Lipid analysis. Hepatic and plasma lipids were measured using the Technicon Axon system (Miles), with the Bayer Triglyceride enzymatic kit, the Bayer Cholesterol enzymatic kit, the free fatty acids NEFA C kit (Wako Chemicals), and the liquid N-geneous HDL cholesterol kit (Instruchemie). To study fat deposits, frozen sections from the livers were cut, stained with a filtered Scharlach red solution (Krajan’s modification), and analyzed by light microscopy using an interactive measurement system (AnalySIS pro).

    Fecal sterols. Feces were collected for 7 d after acclimation of the rats to the experimental conditions and diets. NaBH (2 mL) in ethanol (1 g/L) was added to 0.1 g of powdered dry feces, and fecal total bile acids were prepared according to Suckling et al. (27) with the following modification: neutral sterols were extracted from the samples with n-hexane (2 consecutive washings) before the samples were hydrolyzed. The bile acids were air-dried at 45°C and resolved in 1 mL of isopropanol. Total bile acids were determined enzymatically using a total bile acid diagnostic kit (Sigma 450A) on the Technicon Axon system.

    Amino acids. Amino acids in the diets were determined after hydrolysis in 6 mol/L HCl at 110 ± 2°C for 22 h and prederivatization with phenylisothiocyanate according to the method of Cohen and Strydom (28).

    Fatty acid composition. Fatty acids were extracted from the plasma and liver samples with 2:1 chloroform:methanol (v:v) (29). The samples were filtered, saponified, and esterified in 12% BF3 in methanol (v:v). The fatty acid composition of total lipids from liver and plasma was analyzed as described by Lie and Lambertsen (30). Lipids were extracted from the plasma triacylglycerol-rich lipoprotein fraction (31), separated by TLC on silica gel plates (0.25 mm Silica gel 60, Merck), and developed in hexane:diethyl ether:acetic acid (80:20:1, by vol) (32). The spots were scraped off, and BF3-methanol was added for transesterification (33). To remove neutral sterols and nonsaponifiable material, extracts of the fatty acid methyl esters were heated in 0.5 mol/L KOH in an ethanol:water solution (9:1). Recovered fatty acids were then reesterified using BF3-methanol. The methyl esters were analyzed on a GC8000 Top GC (Carlo Erba Instrument), equipped with a flame ionization detector, and a capillary column coated with a highly polar SP2340 phase (Supelco). Quantification of the fatty acids was made with Chrom-Card A/D 1.0 chromatography station (Carlo Erba Instruments), and the methyl esters were identified by comparison with known standards (Larodan Fine Chemicals) using heneicosanoic acid as the internal standard.

    Malonyl-CoA. Malonyl-CoA in liver was measured by reversed-phase HPLC. Frozen liver (0.1 g) was homogenized in ice-cold 1.4 mol/L HClO4 and 2 mmol/L D-dithiothreitol to obtain a 10% (wt/v) homogenate, and then centrifuged at 12,000 x g for 1 min. Ice-cold 3 mol/L K2CO3 (122 µL) with 0.5 mol/L triethanolamine was added to 500 µL of the supernatant. After 10 min on ice, the solution was centrifuged at 12,000 x g for 1 min at 4°C. The supernatant (40 µL) was injected onto the HPLC, and malonyl-CoA was measured according to Demoz et al. (34), with the following modifications: elution buffer A was adjusted to pH 5.00, and the profile of the gradient elution was as follows: 0 min, 83.5% A; 10 min, 55% A; 17 min, 10% A with a flow-rate of 1.0 mL/min.

    Isolation of plasma triacylglycerol-rich lipoprotein. Plasma from 2 rats was pooled to obtain a volume of 3 mL. The plasma triacylglycerol-rich lipoprotein fraction was prepared as previously described (35).

    Real-time quantitative RT-PCR. Total RNA was purified using Trizol (Gibco BRL) and reversed-transcribed using a Reverse Transcriptase kit (Applied Biosystems). The primers and the Taqman probe for rat {Delta}6 and {Delta}5 desaturases and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were designed using Primer Express (Applied Biosystems); the sequences are listed in Table 3. GAPDH and 18S rRNA were used as endogenous controls. The primers and the Taqman probe for 18S rRNA were purchased from Applied Biosystems. Real-time PCR was carried out in triplicate for each sample on an ABI 7900 sequence detection system (Applied Biosystems) using the protocol generally recommended by Applied Biosystems. For each sample, results were normalized to GAPDH and 18S rRNA. Only results normalized to GAPDH are shown, but these were not different from the results normalized to 18S rRNA.


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  		TABLE 3 Primer sequences used in mRNA quantification by real-time RT-PCR

 
    Statistical analysis. The results are reported as means ± SEM, n = 4–6 rats/group. Statistical analysis was by one-way ANOVA and Dunnett’s test; rats fed the casein-based diet served as controls (Prism, GraphPad).


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
    Plasma and hepatic lipids. In Wistar rats fed soy protein, the plasma cholesterol and triacylglycerol levels were significantly lower than in those fed casein (Table 4). Interestingly, in Wistar rats fed FPH, the plasma cholesterol concentration tended to be lower (P = 0.15) than in casein-fed controls. In obese Zucker rats, the plasma cholesterol levels were lowered by both FPH and soy protein feeding (–34 and –49%, respectively) compared with those fed casein (Table 4). In Wistar rats, the HDL cholesterol level was reduced by the soy protein diet (Table 4), and the HDL cholesterol:total cholesterol ratio was slightly but significantly greater in rats fed both the FPH and the soy protein diets (Table 4). In Zucker rats, the HDL cholesterol level was reduced by FPH and soy protein (–21 and –35%, respectively; Table 4), and the HDL cholesterol:total cholesterol ratio was significantly increased 25% by the soy protein diet (Table 4). The plasma concentration of FFA was greater in Zucker rats fed FPH than in those fed casein (Table 4). The hepatic lipid level, measured by Scharlach red staining, was reduced 36 and 64% in Zucker rats fed FPH and soy protein, respectively, compared with those fed casein (Table 4).


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  		TABLE 4 Plasma lipids, hepatic lipids, and fecal bile acids in Wistar or Zucker rats fed FPH, soy protein, or casein1

 
    Hepatic lipid metabolism. CPT-I activity in the postnuclear fraction was slightly greater in Wistar rats fed FPH (P < 0.05) and soy protein (P = 0.06) compared with controls (Table 5). In Zucker rats, CPT-I activity, measured in the mitochondrial fraction, was not affected by the FPH or soy protein diets. In Zucker rats fed soy protein, CPT-II activity increased 32% (Table 5), and HMG-CoA synthase activity increased 46% (Table 5). FPH feeding did not affect CPT-II or HMG-CoA synthase activities. The 50–60% increase in malonyl-CoA concentration in the livers of Zucker rats fed FPH and soy protein (Table 5) was associated with an increase in the activities of the acetyl-CoA carboxylases and fatty acid synthase, i.e., the cytosolic and mitochondrial isoforms of acetyl-CoA carboxylase were increased 80–90% by FPH feeding, and 60 and 140%, respectively, by soy protein feeding (Table 5). Fatty acid synthase increased 55% in the Zucker rats fed FPH (Table 5). ACAT activity was reduced ~30% in Zucker rats fed FPH and tended to be lower (P = 0.13) in those fed soy protein compared with controls (Table 5). HMG-CoA reductase was several-fold greater in Zucker rats fed FPH and soy protein than in those fed casein (Table 5). The total amount of bile acids in feces was increased 72% in Zucker rats fed soy protein compared with casein-fed rats (Table 4). Interestingly, FPH feeding did not affect the fecal excretion of bile acids.


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  		TABLE 5 Hepatic enzyme activities, hepatic mRNA levels, and hepatic malonyl-CoA concentration measured in Wistar or Zucker rats fed FPH, soy protein, or casein1

 
    Fatty acid composition and desaturases. The relative hepatic amounts of the saturated 14:0 and 16:0 fatty acids were decreased, whereas that of 18:0 was increased in Zucker rats fed FPH and soy protein compared with those fed casein (Table 6). The hepatic level of monounsaturated fatty acids was decreased in Zucker rats fed FPH and soy protein, resulting in a significant reduction in the 18:1(n-9):18:0 ratio in these rats. The hepatic mRNA level of {Delta}9 desaturase, however, was not affected by the dietary proteins (data not shown). Of the (n-6) fatty acids, the level of 18:3(n-6) was unaffected in liver, that of 18:2(n-6) was slightly greater, and those of 20:3(n-6) and 20:4(n-6) were increased several-fold in rats fed the FPH and soy protein diets relative to casein. This resulted in a decreased 18:3(n-6):18:2(n-6) ratio, which was associated with a lower expression of hepatic {Delta}6 desaturase mRNA (Table 5). The 20:4(n-6):20:3(n-6) ratio tended to be lower (P = 0.07 and 0.11) in liver, in accordance with a lower hepatic {Delta}5 desaturase mRNA level (Table 5), whereas the 20:4(n-6):18:2(n-6) and 20:4(n-6):18:3(n-6) ratios were significantly greater in the livers of Zucker rats fed the FPH and soy protein diets. The total level of (n-3) fatty acids in liver was greater in both FPH- and soy protein–fed rats.


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  		TABLE 6 Fatty acid composition in liver of Zucker rats fed FPH, soy protein, or casein1

 
Compared with the fatty acid composition in liver, the fatty acid profile was generally the opposite in plasma (Table 7). In plasma, the relative amounts of the saturated fatty acids 14:0 and 16:0 were increased by FPH and soy protein feeding, whereas the amount of 18:0 was lower after soy protein feeding. The levels of the monounsaturated fatty acids 18:1(n-9) and 16:1(n-7) were increased by FPH and soy protein feeding, resulting in an elevated 18:1(n-9):18:0 ratio. The plasma amount of 18:2(n-6) was higher in rats fed FPH and soy protein, whereas that of 20:4(n-6) was lower, resulting in significantly reduced 20:4(n-6):18:2(n-6) and 20:4(n-6):20:3(n-6) ratios in FPH- and soy protein–fed rats compared with those fed casein. In addition, the plasma 18:3(n-6):18:2(n-6) (P = 0.06) and 20:4(n-6):18:3(n-6) (P = 0.10) ratios tended to be lower in rats fed soy protein than in those fed casein. In the triacylglycerol-rich lipoprotein fraction, the fatty acid composition generally resembled the plasma profile (Table 8), i.e., the amount of 18:2(n-6) increased and the amount of 20:4(n-6) decreased, resulting in reduced 20:4(n-6):18:2(n-6), 20:4(n-6):18:3(n-6) and 20:4(n-6):20:3(n-6) ratios in rats fed FPH and soy protein. In plasma, the level of 18:3(n-3) was increased by FPH and soy protein feeding, and that of 20:5(n-3) was increased significantly only in FPH-fed rats. The level of 22:6(n-3) was reduced by both FPH and soy protein feeding. In the triacylglycerol-rich lipoprotein fraction, the composition of the (n-3) fatty acid pattern was affected only in phospholipids, similar to the findings in plasma.


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  		TABLE 7 Fatty acid composition in plasma of Zucker rats fed FPH, soy protein, or casein1

 

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  		TABLE 8 Fatty acid composition in the different lipid classes in the triacylglycerol-rich lipoprotein fraction of Zucker rats fed FPH, soy protein, or casein1

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
A number of studies using different animal models have demonstrated the hypocholesterolemic effects of soy protein. In the present study, these effects were compared with that of an enzymatically hydrolyzed salmon protein (FPH). First, a study in male Wistar rats was performed. In these rats, soy protein lowered the plasma lipids (Table 4) and FPH feeding tended to lower plasma lipids. However, the ratio of HDL cholesterol to total cholesterol (Table 4) as well as the activity of the mitochondrial ß-oxidation enzyme, CPT-I, was significantly increased in Wistar rats fed FPH (Table 5), suggesting that FPH may affect lipid metabolism. On the basis of these results, we investigated the effects of FPH on lipid metabolism in more detail in a hyperlipidemic animal model, i.e., the obese (fa/fa) Zucker rat (8).

In Zucker rats fed FPH and soy protein, the plasma concentration of cholesterol was lower compared with rats fed casein (Table 4). Several mechanisms may explain the hypocholesterolemic effect of these dietary proteins. Enhanced fecal steroid excretion (36), which is the major route for cholesterol excretion from the body, may be 1 mechanism that explains the cholesterol-lowering effect of soy protein. In the present Zucker rat experiment, soy protein, but not FPH, increased the excretion of fecal bile acids (Table 4). Therefore, in the soy protein–fed rats, the hypocholesterolemic effect may be due at least in part to elevated excretion of bile acids in feces. In contrast, dietary FPH did not affect the fecal bile acid excretion compared with dietary casein; thus, another mechanism must be responsible for the cholesterol-lowering effect of FPH. It was suggested that soy protein compensates for the fecal loss of steroids by stimulating HMG-CoA reductase activity, the rate-limiting enzyme of cholesterol biosynthesis (37). In the present experiment, both FPH and soy protein increased the hepatic activity of HMG-CoA reductase (Table 5). Thus, changes in HMG-CoA reductase activity may be a compensatory response to the reduced concentration of cholesterol in plasma, and not to the increased excretion of fecal steroids.

ACAT catalyzes the reaction in which fatty acyl-CoA is esterified with cholesterol. Cholesteryl ester may then be stored in the cytoplasm as lipid droplets or be secreted as part of VLDL along with free cholesterol. Thus, ACAT plays a major role in VLDL secretion and the subsequent cholesteryl ester accumulation and increased risk of cardiovascular disease (38). In the present Zucker rat experiment, ACAT activity was significantly decreased by FPH feeding compared with casein feeding (Table 5). Because there is strong evidence that increased ACAT activity plays an important role in the progression of atherosclerosis (39,40), this finding indicates that FPH is cardioprotective and is involved in the regulation of plasma cholesterol.

A potential explanation for the cholesterol-lowering effect of FPH and soy protein lies in their amino acid profiles, especially methionine, glycine, lysine, and arginine. The methionine content of the soy diet was less than half that of the casein diet, whereas the methionine concentration of the FPH diet was intermediate (Table 2). Methionine was shown to elevate serum cholesterol concentration (41). However, methionine supplementation to a soy diet did not abolish the hypocholesterolemic effects of soy protein relative to casein (42), suggesting that some factor other than methionine may be responsible at least in part for the cholesterol-lowering effect of soy protein. However, it was suggested that the higher ratio of methionine to glycine in casein may be responsible for the elevation in serum cholesterol (43), and glycine supplementation to a casein-based diet lowered the serum cholesterol concentration in rats (41). In the present experiment, the glycine concentration in the FPH and soy protein diets was higher than that of the casein diet (Table 2), yielding a 80–85% lower methionine:glycine ratio in the FPH and soy protein diets (Table 2). Thus, the glycine content of the dietary proteins may cause the differences found in the plasma cholesterol level of the current study. It was suggested that the increased serum cholesterol level that occurs with casein feeding is caused by the high ratio of lysine to arginine in casein (43,44). The dietary lysine:arginine ratios in the current study were 1.8 in the case of casein, and 1.1 and 0.8 in the case of FPH and soy protein, respectively (Table 2), favoring a cholesterol-lowering effect by FPH and soy protein. Thus, the amino acid composition of FPH and soy protein may be responsible at least in part for the effect of the protein source on plasma cholesterol levels.

The fatty acid profile in plasma and liver differed between Zucker rats fed the various experimental diets. The increased hepatic level of 18:0 and the correspondingly decreased level of 18:1(n-9) in rats fed FPH and soy protein compared with those fed casein reduced the ratio of 18:1(n-9) to 18:0 (Table 6), which is an indirect index of {Delta}9 desaturase activity. The gene expression of {Delta}9 desaturase was unchanged, however (data not shown), indicating that the change in fatty acid composition was not due to transcriptional regulation of {Delta}9 desaturase activity. The lower 18:3(n-6):18:2(n-6) and 20:4(n-6):20:3(n-6) ratios (Table 6), indices of {Delta}6 desaturase and {Delta}5 desaturase activities, respectively, observed in the rats fed FPH and soy protein diets suggest a lower rate of desaturation. Because the mRNA levels of {Delta}5 and {Delta}6 desaturase were reduced (Table 5), the data argue for decreased activities of {Delta}5 and {Delta}6 desaturases after FPH and soy protein feeding. The hepatic ratio of 20:4(n-6):18:2(n-6) is often used as an index of {Delta}5 desaturase, and it was observed in rats that casein promotes this metabolism of linoleic acid to arachidonic acid more than does soy protein (45). In contrast, the present experiment showed an increased 20:4(n-6):18:2(n-6) ratio when the rats were fed FPH and soy protein compared with when the casein diet was fed (Table 6). The opposite fatty acid profile in liver and plasma may be due to a specific secretion of glycerolipids in the triacylglycerol-rich lipoproteins because the major changes in the plasma fatty acid profile were also seen in the triacylglycerol-rich lipoprotein fraction (Tables 7, and 8). Zucker rats fed FPH and soy protein likely had a higher secretion of triacylglycerol-rich lipoproteins from the liver than rats fed casein. This is indicated by the morphological data showing a higher lipid content in the livers of rats fed casein than in those fed FPH or soy protein (Table 4), and also by the high hepatic (n-6):(n-3) ratio and the low hepatic 20:4(n-6):18:2(n-6) and [20:5(n-3)+22:6(n-3)]:18:3(n-3) ratios in rats fed casein (calculated from Table 6), which indicates hepatic steatosis (46).

It was found that 20:5(n-3) stimulates mitochondrial ß-oxidation, whereas 22:6(n-3) is more effective for peroxisomal ß-oxidation (13); it is possible that the elevated concentrations of these fatty acids in livers of Zucker rats fed FPH and soy protein (Table 6) may have affected the activities of mitochondrial and peroxisomal ß-oxidation enzymes. In agreement with an increased level of hepatic 22:6(n-3) in Zucker rats fed FPH and soy protein, the peroxisomal ß-oxidation enzyme fatty acyl-CoA oxidase was upregulated (unpublished data). CPT-I is normally the regulating enzyme of mitochondrial ß-oxidation, but there was no change in this enzyme in Zucker rats fed FPH or soy protein (Table 5). This does not exclude an effect on mitochondrial ß-oxidation because CPT-II was recently suggested to be the regulating enzyme of mitochondrial ß-oxidation when the influx of fatty acids to the liver is high (47). Under these circumstances, CPT-II activity is highly correlated with HMG-CoA synthase activity. It was therefore of interest to find that both HMG-CoA synthase activity and CPT-II activity increased in Zucker rats fed soy protein (Table 5), arguing for elevated mitochondrial ß-oxidation with the subsequent synthesis of ketone bodies in these soy protein–fed rats. The unchanged CPT-I activity may be due to a high hepatic concentration of malonyl-CoA, the product of acetyl-CoA carboxylase, in rats fed FPH and soy protein (Table 5). Abu-Elheiga et al. (48) suggested that the mitochondrial form of acetyl-CoA carboxylase is involved in the regulation of mitochondrial fatty acid oxidation through inhibition of CPT-I, the regulating enzyme of mitochondrial ß-oxidation, by its product, malonyl-CoA, whereas the cytosolic form is involved in fatty acid synthesis. Indeed, induction of the cytosolic form of acetyl-CoA carboxylase tended to be higher after feeding FPH than soy protein (P = 0.051); this is in accord with the increased activity of the lipogenic enzyme fatty acid synthase (Table 5).

In summary, although the effect of soy protein on lipid metabolism generally was slightly more pronounced than that of FPH, the cardioprotective effect of FPH was clearly demonstrated. The mechanism for the cholesterol-lowering effect of FPH is different from that of soy protein because FPH lowered plasma cholesterol in Zucker rats by reducing the activity of ACAT, whereas soy protein did so by also affecting the secretion of fecal bile acids.


   ACKNOWLEDGMENTS
 
Svein Kruger, Kari H. Mortensen, Randi Sandvik, Randi Solheim, Liv-Kristine Øysæd, Laila Vårdal, Karen Böhm-Nilssen, and Thu Thao Nguyen are acknowledged for their technical assistance. Aase Heltvedt is especially thanked for taking care of the experimental rats. Rolf Olsen Skarholmen (Askøy, Norway) provided fresh salmon frames, and Novozymes AS (Denmark) provided Protamex and facilities for the production of FPH.


   FOOTNOTES
 
1 The research was supported by Hordafor AS (Norway) and the Norwegian research council. Back

3 These authors contributed equally to this work. Back

4 Abbreviations used: ACAT, acyl-CoA:cholesterol acyltransferase; CPT, carnitine palmitoyltransferase; CVD, cardiovascular disease; FPH, fish protein hydrolysate; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HMG, 3-hydroxy-3-methylglutaryl; NIDDM, noninsulin-dependent diabetes mellitus. Back

Manuscript received 26 November 2003. Initial review completed 10 January 2004. Revision accepted 25 February 2004.


   LITERATURE CITED
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 

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