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Original Research Paper |
Beltsville Human Nutrition Research Center, Agricultural Research Service, U.S. Department of Agriculture, Beltsville (S.J.B.)
National Institutes of Health, Animal Genetic Resource, Bethesda (C.T.H.)
The Jerome Holland Laboratory, Department of Experimental Pathology, American Red Cross, Rockville (C.H.), Maryland
Department of Medicine, George Washington University Medical Center, Washington DC (P.L., T.R., M.T.V.)
Virginia State University, Petersburg, Virginia (A.I.M.)
Department of Food Science, Ain Shams University, Cairo, EGYPT (A.A.A.)
Address reprint requests to: Sam J. Bhathena, PhD, Phytonutrients Laboratory, Beltsville Human Nutrition Research Center, Bldg. 307, Room 315, Beltsville, MD 20705. E-mail: bhathens{at}ba.ars.usda.gov
ABSTRACT
Objective: Soy protein and flaxseed meal have been reported to have beneficial effects on many chronic diseases in humans and animals. The primary objective of the study was to evaluate the beneficial effects of soy protein and flaxseed meal on hypertriglyceridemia and liver steatosis associated with obesity and diabetes. We compared the effects of dietary soy protein and flaxseed meal with that of casein on plasma and liver lipids in a genetic model of obesity, type II diabetes and insulin resistance, namely the SHR/N-cp rat.
Methods: Lean and obese phenotypes of SHR/-cp rats were fed AIN 93 diets containing 20% of energy from casein (control), soy protein concentrate or flaxseed meal for six months. Plasma was analyzed for total cholesterol, LDL cholesterol, triglyceride and total protein. Liver was analyzed for steatosis by light microscopy after staining samples with Hematoxylin-Eosin and Oil-Red-O.
Results: In lean rats soy protein and flaxseed meal significantly decreased plasma total cholesterol (26.0% and 20.3% respectively) compared to casein. In obese rats flaxseed meal had significant cholesterol lowering effect compared to control rats (41%). Soy protein significantly lowered both plasma LDL-cholesterol and HDL-cholesterol in lean phenotypes while in obese phenotypes flaxseed meal significantly lowered LDL-cholesterol and HDL-cholesterol compared to casein-fed rats. Flaxseed meal also significantly lowered plasma triglyceride in both lean and obese rats compared to casein fed rats (33.7% and 37% respectively). There was significantly greater fat accumulation in livers of obese rats than lean rats (200%) regardless of dietary protein type. Flaxseed meal significantly lowered fat deposition in livers of both lean and obese rats compared to rats fed casein or soy protein. Dietary component(s) present in flaxseed meal or soy protein responsible for hypolipidemic effects is not clear.
Conclusions: The marked hypotriglyceridemic and hypocholesterolemic effects of flaxseed meal may have important therapeutic implications in patients with hypertriglyceridemia and hypercholesterolemia and deserve further study in humans with these disorders. Flaxseed meal supplementation may provide a new therapeutic strategy to reduce hypertriglyceridemia and fatty liver.
Key words: cholesterol, flaxseed, fatty liver, hypertriglyceridemia, plasma lipids, soy protein
INTRODUCTION
In recent years, soy foods have attracted a great deal of interest among consumers and health care professionals for their potential benefits on human health. This is based on accumulated data from epidemiologic as well as nutritional intervention studies in humans and animals suggesting that consumption of soy and soy-based products have protective effects on a variety of chronic diseases, especially cardiovascular disease [1–5]. Indeed, numerous studies in humans and animals have shown that consumption of soy protein reduces serum total cholesterol and low-density lipoprotein (LDL) cholesterol, which are the major risk factors of cardiovascular disease [6–12]. This evidence has led to the approval by the United States Food and Drug Administration of a health claim that "consumption of 25 g of soy protein a day, as part of a diet low in saturated fat and cholesterol, may reduce the risk of heart disease"[13]. More recently, the American Heart Association (AHA) Nutrition Committee affirmed that the use of soy foods was consistent with the AHA dietary guidelines but recommended that more research on the effects of soy protein and related phytochemicals on blood lipids be carried out [14].
Besides soybean, other plant seeds, particularly flaxseed, have also received increasing attention for their potential role in preventing lipid disorders. However, relatively few data are available regarding the impact of flaxseed on blood lipids. Some studies have shown that whole flaxseed lower serum cholesterol in animals and humans [15–18], but other studies do not show such an effect [19]. Since whole flaxseed is difficult to digest, it is possible that studies not showing an effect may have used whole seed rather than flaxseed meal. Studies comparing the nutritional effects of these two dietary sources of plant protein and their impact on serum lipids and other cardiovascular risk factors are lacking.
The spontaneously hypertensive/NIH-corpulent (SHR/N-cp) rat is a genetic animal model that exhibits obesity, type II diabetes mellitus and mild hypertension [20–23]. The SHR/N-cp rat is congenic and was originally derived by an initial mating of a male obese spontaneously hypertensive (Koletsky) rat which was heterozygous for the cp gene with a spontaneously hypertensive rat (SHR) of the Okamoto strain, followed by multiple cycles of back-crossing of the progeny to the SHR strain [20, 21]. A minimum of ten backcrosses was carried out to eliminate the non-cp genes of the Koletsky strain. Obese homozygotes, unlike their lean littermates, exhibit marked obesity, glucose intolerance, hyperinsulinemia, hyperlipidemia and mild hypertension. Obese SHR/N-cp rats also show marked elevations in plasma leptin, which correlate with plasma lipid and insulin levels [24]. A consistent pathologic finding associated with hyperlipidemia in obese SHR/N-cp rats is steatosis in the liver [21], a feature most commonly found in humans with obesity, type II diabetes and the metabolic syndrome X [25–28].
The role of dietary soy protein and flaxseed on hyperlipidemia and fatty liver change in this strain has not been examined. We investigated the effects of dietary soy protein concentrate and flaxseed meal on plasma lipids and liver abnormalities including pathological changes in the liver in the SHR/N-cp rat.
METHODS
Animals
Male lean and obese SHR/N-cp rats were obtained from the National Institutes of Health at approximately five to six weeks of age. At this age, obesity is already evident in SHR/N-corpulent (cp/cp) rats as indicated by higher body weight (average 125 g) than their lean littermates (average 96 g) and increased abdominal girth. The experimental protocol was approved by the Institutional Animal Care and Use Committees of the Agricultural Research Service, U.S. Department of Agriculture, Beltsville, Maryland, and by the George Washington University, Washington, D.C. All animals were housed individually in stainless steel wire cages with controlled temperature (21° to 25°C) and relative humidity (40% to 50%) and maintained on a reverse 12-hour dark (0900 to 2100 hours) and light (2100 to 0900 hours) cycle.
Diets and Experimental Protocol
All animals were provided with a Purina rat chow and were maintained on this diet for two weeks until seven to eight weeks of age. Food and water were consumed ad libitum. The rats were then randomly divided into three groups of 10 lean rats and three groups of 10 obese rats and fed AIN-93 diet [29] supplemented with casein, soy protein concentrate or flaxseed meal, as the sole source of dietary protein. Group 1 rats received 20% of energy from casein; group 2 rats received 20% of energy from soy protein, and group 3 rats received 20% of energy from flaxseed meal. Soy protein concentrate and flaxseed meal were analyzed for isoflavones and lignans by Archer Daniels Midland company. Soy protein concentrate contained 234.9 mg/kg total isoflavones. Flaxseed meal contained 17.0 mg/kg secoisolariciresinol diglucoside. With the exception of the protein source, all three diets were identical and contain similar amounts of protein, fat, carbohydrates, minerals, and vitamins. All diets contained (g/kg) dextrinized cornstarch, 155; sucrose, 100; corn oil, 40; cellulose, 50; mineral mix (AIN-93M-MX), 35; vitamin mix (AIN-93-VX), 10; L-cystine, 1.8; choline bitartrate, 2.5; and tert-butylhydroquinone, 0.008. Amount of corn starch was adjusted in each diet to provide 20% of energy from protein. Casein and L-cystine were purchased from Sigma Chemicals, St. Louis, MO. Soy protein concentrate and flaxseed meal were obtained gratis from Protein Specialties Division, Archer Daniels Midland, Decatur, IL. Tert-butylhydroquinone was purchased from Aldrich Chemical Co., Milwaukee, WI. All other ingredients were purchased from Dyets Inc., Bethlehem, PA.
All animals were fed the experimental diets for six months and weighed biweekly throughout the study. Food intake was measured biweekly over two-day period. At the end of the feeding period, animals were killed by decapitation under carbon dioxide anesthesia. Blood was collected in tubes containing EDTA (1.4 g/L) and Trasylol (100 kU/L), and plasma was separated for subsequent biochemical analyses. Immediately after death, the abdomen was opened by a midline incision and the liver was quickly removed and weighed and a representative sample of liver tissue was excised, frozen in liquid nitrogen and processed for histopathological examination.
Analytical Measurements
Plasma levels of total cholesterol and HDL cholesterol were measured enzymatically using Alcyon analyzer, ATAC 8000 (Abbott Laboratories) using kits from Elan Diagnostics (cat. #s. 516-018 and 541-235 respectively) while triglyceride and total protein were measured using the same equipment and kits from Biochem Lab Systems (cat. #s. 589-008 and 582-008). LDL cholesterol was calculated by subtracting HDL cholesterol from total cholesterol.
Histology
Sections of snap frozen liver were cut at 3 µ and stained with hematoxylin-eosin (H&E) and oil Red O (ORO) to assess for the presence of lipids. Additional sections were routinely processed for light microscopy with formalin fixation, embedded in paraffin and stained with H&E. Histological evaluation included a semi-quantitative analysis of the presence of micro- and macrovesicular fat. All sections were coded and analyzed blindly by the pathologist without knowledge of related characteristics or diet. The degree of fat accumulation was graded on a scale of 0 to 4 at intervals of 0.5 as follows: 0 = no evidence of or barely visible microvesicular fat, 1+ = <25%; 2+ = 25% to 49%, 3+ = 50% to 75% and 4+ = fat involving >75% of lobule. Half-grades were established by ranking of slides within each full grade.
Statistical Analysis
Results are expressed as mean ± standard error of the mean. Comparisons between groups for the data in Tables 1 and 2 were made using one-way analysis of variance (ANOVA) and for the data in Table 3 by two way ANOVA. When an effect was statistically significant (p < 0.05) mean comparisons were done. A Sidak adjusted significance level was used for the pair-wise comparisons of the means so that the overall significance level was 0.05.
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The results of experiments performed in lean and obese SHR/N-cp rats fed the three different protein diets are summarized in Tables 1 and 2, respectively. The mean food intake by lean rats was 22.8 g/day while that of obese rats was 28.9 g/day. There were no significant differences in food intake between the three diets (data not shown).
Lean SHR/N-cp Rats
At the end of six months of feeding, lean rats had similar mean body weights and weight gain, regardless of the source of the dietary protein. No significant differences were observed for mean liver weights (absolute or relative to body weight) in lean rats fed different diets. Plasma total protein concentrations were slightly lower in rats fed flaxseed meal but not soy protein, compared to rats fed casein. However, the decrease was not statistically significant. Plasma total cholesterol was significantly lower in rats fed flaxseed meal and soy protein than in those fed casein, while HDL-cholesterol and LDL-cholesterol levels were significantly lower only in rats fed soy protein compared to casein. Plasma triglyceride concentration was significantly lower in rats fed flaxseed meal than in those fed soy protein or casein. There was no significant difference in triglyceride concentration between soy protein-fed and casein-fed animals.
Obese SHR/N-cp Rats
Compared to lean rats, obese rats had distinctly higher body weight, weight gain and liver weight (absolute and relative to body weight) and exhibited markedly elevated plasma concentrations of total cholesterol, HDL-cholesterol, LDL cholesterol and triglyceride, but had similar levels of plasma total protein. After six months of feeding, liver weights were significantly lower in rats fed flaxseed meal than those fed casein or soy protein concentrate. Liver weights of rats fed soy protein was also lower than those fed casein but the differences were not significant. Liver weights as percentage of body weight were significantly lower in rats fed flaxseed meal than in rats fed casein. Relative liver weights of rats fed soy protein concentrate were not significantly different from those of rats fed either casein or flaxseed meal. Plasma total protein concentrations were significantly lower in obese rats fed flaxseed meal and soy protein than those fed casein. Similar to findings in lean rats, total plasma cholesterol, HDL-cholesterol, LDL cholesterol and triglyceride levels were significantly lower in obese rats fed flaxseed meal than in those fed casein. Total plasma cholesterol, LDL cholesterol and triglyceride levels were also lower in obese rats fed soy protein compared to those fed casein, but the differences were not statistically significant. Plasma triglyceride concentration was reduced by 37% in rats fed flaxseed meal and by 19% in rats fed soy protein compared to those fed casein.
Liver Histology
Fig. 1 shows the ORO-stained sections of livers from lean and obese rats fed casein, soy protein or flaxseed meal. Regardless of type of diet or phenotype, small amounts of fat accumulation were observed in the liver as microvesicles of fat (vesicles estimated at less than 15 µ) within periportal hepatocytes. Livers from lean rats showed minimal or barely visible fat in hepatic cells as demonstrated by the sparse distribution of small microvesicles within hepatocytes. By contrast, livers from obese rats showed widespread deposition of fat globules of different sizes inside parenchymal cells. The number of microvesicles and the extent of lobular involvement appeared to increase in parallel at grades of 0.5 to 1+ or greater. Macrovesicles of fat (vesicles greater than 15 µ) appeared to localize to both pericentral and periportal hepatocytes with a slight preponderance in pericentral zones, except at the highest grades of fat accumulation.
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DISCUSSION
The present study demonstrates for the first time that substitution of flaxseed meal or soy protein for casein in the diet significantly reduces fat accumulation in the liver of obese SHR/N-cp rats with marked hyperlipidemia. In addition, the more striking finding in this study is that flaxseed meal as compared to soy protein produced a more pronounced reduction of liver weight and hepatic fat deposition in obese animals. Regardless of type of diet, obese rats, in contrast to their lean littermates, exhibited marked hepatomegaly with diffuse accumulation of fat in liver parenchyma, as has been observed in previous study [21]. Histologically, livers of lean rats showed a minimal amount of fat, appearing as sparse microvesicles (size of <15 µ) localized in periportal hepatocytes. By contrast, livers from obese rats showed a more extensive deposition of fat globules of different sizes, both microvesicles and macrovesicles (size of >15 µ) widely distributed throughout the hepatic lobule. The fatty liver seen in obese SHR/N-cp rats closely resembles the pathologic features of nonalcoholic fatty liver disease (NFLD) described in humans with obesity, type II diabetes mellitus and metabolic syndrome X [25–28], conditions which, like the obesity in the SHR/N-cp rat, have in common the metabolic features of peripheral insulin resistance. Of note in the present study is that flaxseed meal but not soy protein induced significant reductions of hepatic fat even in lean animals with lower grades of fatty deposition, suggesting that the antilipogenic effects of flaxseed meal on the liver is independent of obesity. The beneficial effects of flaxseed meal and soy protein on fatty liver did not appear to be related to differences in body weight gain, since there were no significant differences in weight gain among the three diet groups. This was true for both the lean and obese rats. It should be noted that, with the exception of the protein source, the diets were identical in the total amounts of protein, fat, carbohydrate, minerals and vitamins. Further, the food intakes were similar in rats fed casein, soy protein concentrate or flaxseed meal. Therefore, it seems unlikely that the different effects of flaxseed meal and soy protein on hepatic fatty deposition can be ascribed to differences in energy or fat intake.
The other striking finding in the present study is that flaxseed meal supplementation also produced substantial reductions in triglyceride concentration in both lean and obese rats, whereas soy protein ingestion had minimal or no significant effects on triglyceride levels in the two groups of animals. Thus, flaxseed meal has a greater hypotriglyceridemic effect than soy protein, independently of the level of triglyceridemia. Crouse et al. [30] also observed no significant effect of soy protein on plasma triglycerides in healthy human subjects.
There are numerous reports documenting the hypocholesterolemic effect of soy protein [6–12], but only a few studies have examined the effects of flaxseed on serum lipids in animals and humans. Ratnayake et al. showed that a 20% and higher flaxseed diet given for 90 days in rats decreased serum total cholesterol [15]. However, Babu et al. obtained different results in their studies of young female Sprague-Dawley rats [19]. These investigators fed isoenergic modified AIN diet supplemented with either whole ground flaxseed or defatted flaxseed meal to these animals for 56 days but found no significant effect on total cholesterol. In addition, plasma triglyceride was increased, if at all, by defatted flaxseed meal, but was not changed by ground flaxseed. In studies of hypercholesterolemic rabbits, Prasad showed that dietary flaxseed reduced total and LDL cholesterol and prevented hypercholesterolemic atherosclerosis [16]. Cunnane et al. showed that consumption of 50 grams of flaxseed/day for four weeks resulted in a small but significant reduction in LDL cholesterol in young healthy humans [17]. Bierenbaum et al. showed that flaxseed supplementation in the form of either a flaxseed-containing bread or 15 g of ground flaxseed for three months resulted in significant reductions in serum total and LDL cholesterol with no change in HDL cholesterol in human subjects with hyperlipidemia [18]. Similar results were obtained by Jenkins et al. with partially defatted flaxseed in hypercholesterolemic subjects without obesity and diabetes [31]. The results of these studies suggest that the effects of flaxseed may vary according the experimental models, the amount or type of flaxseed preparation and the pre-existing level of serum lipids. Unfortunately, serum triglyceride levels were not measured in hyperlipidemic subjects in these studies. Moreover, some of these studies have used either ground flaxseed or partially defatted flaxseed, which may have confounded the results. In the present study flaxseed meal also lowered HDL-cholesterol in obese rats compared to casein. The decrease in HDL-cholesterol in lean rats was not significant.
The components in soy protein concentrate or flaxseed meal responsible for their salutary effects on plasma lipids and fatty liver have not been fully identified. Whole soybeans and flaxseeds are both rich in proteins and fat. Soybeans, unlike most other beans, contain relatively high amounts of fat (about 47%) and protein (about 36%) [32, 33]. The predominant fatty acid in soybeans is linoleic acid, although a lesser amount of n-3 fatty acid, alpha-linolenic acid, is also found. Linoleic acid has been reported to lower cholesterol. The ratio of linoleic to alpha-linolenic acid in soybeans is about 7.5:1 [33]. Whole flaxseed contains approximately 41% fat and 21% protein of the seed weight [34]. Unlike soybean, flaxseed is particularly rich in alpha-linolenic acid (approximately 57% of the total fatty acids in flaxseed), which has lipid lowering properties [34,35]. Thus, the reduction of blood cholesterol by either dietary soy protein or flaxseed in these studies may be due in part to linoleic acid and alpha-linolenic acid present in whole bean or seed. However, both soy protein concentrate and flaxseed meal used in the present study, unlike whole soybean and whole ground flaxseed, have been fully defatted and do not contain an appreciable amount of linoleic or alpha-linolenic acid. Therefore, it is highly unlikely that these fatty acids were responsible for the observed reduction of plasma cholesterol and triglyceride that was associated with soy protein or flaxseed meal in the present study. Our results obtained with defatted flaxseed meal in obese SHR/N-cp rats extend the recent observations of Prasad et al., who showed that a diet with a CDC-flaxseed (type II flaxseed) with very low alpha-linolenic acid content reduces total serum cholesterol and LDL cholesterol in rabbits [36].
Other major constituents of soy protein and flaxseed which may be responsible for their lipid lowering effects are the phytoestrogens, namely isoflavones and lignans. Soy protein isolate is a rich source of isoflavones genistein and daidzein [37,38], which exert hypocholesterolemic effects in animals and humans [30, 39–44]. Isoflavones may have contributed to the hypolipidemic effects of soy protein concentrate used in the present study which contained 234.9 mg/kg total isoflavones. Flaxseed is the richest source of lignans [45], which have also been reported to have antioxidant and hypolipidemic effects [46,47]. Indeed, secoisolariciresinol diglucoside (SDG), a major lignan isolated from flaxseed, has been shown to reduce serum total and LDL cholesterol in hypercholesterolemic rabbits [47]. The amount of SDG in the flaxseed meal used in this study was 17 mg/kg. Whether SDG has an effect on serum triglyceride remains to be determined.
The mechanisms by which soy protein and flaxseed meal reduce plasma lipids and fatty accumulation in liver are unclear. One of the several possible mechanisms that have been proposed for hypocholesterolemic effects of soy protein is that an increase in bile acid excretion induced by soy protein ingestion enhances removal of LDL, increases free thyroxine secretion and alters hepatic metabolism in a way that augments LDL removal by hepatocytes, and increases LDL receptors [48]. Whether flaxseed has a similar effect on bile acid secretion, hepatic cholesterol metabolism or LDL receptors is not known. Hypertriglyceridemia is a characteristic lipid abnormality in obesity and type II diabetes mellitus and has been linked to the development of fatty liver in obesity [49,50]. In obesity, accumulation of triglycerides in the liver has been suggested to occur in response to an increased flux of fatty acids to the liver from dietary sources, from adipose tissue or from increased endogenous synthesis of fatty acids [51]. It is reasonable to suggest that the improvement of fatty liver by flaxseed supplementation shown in our studies in SHR/N-cp rats may be related to the substantial reduction of plasma triglyceride induced by flaxseed meal in these animals. The reduction in total and relative liver weight in rats fed flaxseed meal compared to those fed casein may in part be due to reduced fat accumulation.
In summary, we have shown for the first time that dietary supplementation with flaxseed meal and to a lesser extent soy protein concentrate markedly improved both liver steatosis and plasma hypercholesterolemia and hypertriglyceridemia in obese SHR/N-cp rats. Flaxseed meal supplementation in these animals produced a more pronounced effect in reducing fat accumulation in liver. Flaxseed meal supplementation also appeared to reduce plasma triglyceride levels to a greater extent than soy protein in both phenotypes. It is not clear which dietary component(s) present in flaxseed meal or soy protein is (are) responsible for the observed hypolipidemic effects. The marked hypotriglyceridemic and hypocholesterolemic effects of flaxseed meal may have important therapeutic implications in patients with hypertriglyceridemia and hypercholesterolemia and deserve further study in humans with these disorders. Flaxseed meal supplementation may provide a new therapeutic strategy to reduce hypertriglyceridemia and fatty liver.
ACKNOWLEDGMENTS
We thank the Archer Daniels Midland Company, Protein Specialties Division, Decatur, IL. for the generous gift of soy protein and flaxseed meal and the analysis of isoflavone and lignan content of these products. We also thank Mary J. Camp, Biometrical Consulting Service, Agricultural Research Service, for the statistical analyses of the data.
Received September 16, 2002. Accepted December 11, 2002.
REFERENCES
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  L. T. Bloedon, S. Balikai, J. Chittams, S. C. Cunnane, J. A. Berlin, D. J. Rader, and P. O. Szapary Flaxseed and Cardiovascular Risk Factors: Results from a Double Blind, Randomized, Controlled Clinical Trial J. Am. Coll. Nutr., February 1, 2008; 27(1): 65 - 74. [Abstract] [Full Text] [PDF]
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 L. Gutierrez, G. Conejero, M. Castelain, S. Guenin, J.-L. Verdeil, B. Thomasset, and O. Van Wuytswinkel Identification of new gene expression regulators specifically expressed during plant seed maturation J. Exp. Bot., June 1, 2006; 57(9): 1919 - 1932. [Abstract] [Full Text] [PDF]
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W.M.N. Ratnayake, G.S. Gilani, S. J. Bhathena, A. A. Ali, C. Haudenschild, P. Latham, T. Ranich, A. I. Mohamed, C. T. Hansen, and M. T. Velasquez Letters to the Editor J. Am. Coll. Nutr., August 1, 2003; 22(4): 326 - 329. [Full Text] [PDF]
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