QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Madani, S. Articles by Belleville, J. Search for Related Content PubMed PubMed Citation Articles by Madani, S. Articles by Belleville, J.

---

Biochemical and Molecular Actions of Nutrients

VLDL Metabolism in Rats Is Affected by the Concentration and Source of Dietary Protein¹

Unité de Nutrition Cellulaire et Métabolique, Faculté des Sciences Gabriel, Dijon, France

²To whom correspondence should be addressed. E-mail: jbellev{at}u-bourgogne.fr.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The present study was designed to determine if changes in dietary protein level and source are related to changes in VLDL lipid concentrations and VLDL binding by hepatic membranes and isolated hepatocytes. Male Wistar rats were fed cholesterol-free diets containing 10, 20 or 30 g/100 g casein or highly purified soybean protein for 4 wk. Hepatic, plasma and VLDL lipids, VLDL apo B-100 and VLDL uptake by isolated hepatocytes and VLDL binding to hepatic membrane were determined. Increasing casein or soybean protein level (from 10 to 30 g/100 g) in the diet increased VLDL apo B-100, indicating an increase in the number of VLDL particles. VLDL uptake by isolated hepatocytes and VLDL binding to hepatic membrane increased when the protein level increased from 10 to 20 g/100 g in the diet and decreased with 30 g/100 g protein, regardless of protein type. The dietary protein source did not affect plasma total cholesterol concentrations at any protein level. Feeding 20 g/100 g soybean protein compared with casein lowered plasma triglyceride concentrations and VLDL number as measured by decreased VLDL-protein, -phospholipid, -triglyceride, -cholesterol and -apo B-100. VLDL uptake by isolated hepatocytes and VLDL binding to hepatic membrane were higher in rats fed soybean protein than those fed casein. The higher VLDL uptake could be responsible for the hypotriglyceridemia in rats fed soybean protein.

KEY WORDS: • soybean protein • lipoproteins • rat • VLDL metabolism • dietary protein

Dietary protein has been shown to affect plasma cholesterol concentration. Indeed, in different experimental species, dietary soybean protein isolate reduces serum cholesterol concentrations compared with casein (1). The substitution of soybean protein for animal protein in the diet reduces the concentrations of serum cholesterol in humans. This effect is somewhat variable but is generally greater in hypercholesterolemic than in normocholesterolemic subjects (2–4). Soybean protein lowers serum triglyceride and apo B levels in humans (5) as well as in experimental animals (6). Triglyceride levels transported principally by VLDL depend on the activities of lipoprotein lipase and the membrane cellular receptor (B/E receptors) (7). Soybean protein and casein consumed by rats do not affect plasma lipoprotein lipase activity (8). The dietary protein source, however, influences the activity of the hepatic lipoprotein receptors that regulate VLDL uptake. Cohn et al. (9) have reported that in rats fed a high cholesterol-casein diet, plasma cholesterol accumulates in the VLDL fraction due to excess VLDL production and inadequate VLDL removal. Sirtori et al. (10) have shown in rats that VLDL binding to hepatic membranes is markedly enhanced by a cholesterol-enriched soybean protein diet compared with a casein diet. These results suggest that in rats fed high cholesterol diets, soybean protein increases the activity and/or the number of specific hepatic VLDL receptors. However, a cholesterol feeding effect on the lipoprotein clearance rate cannot be completely excluded. Indeed, excess cholesterol suppresses cholesterol biosynthesis and leads to the downregulation of hepatic B/E receptors (11). Moreover, it has been reported that an elevated cholesterol ester concentration in triglyceride-rich lipoproteins in rats fed cholesterol decreases the lipolysis rate catalyzed by hepatic lipase that rapidly removes VLDL remnants from circulation (12). Although these studies clearly demonstrate that soybean protein compared with casein accelerates VLDL uptake by liver, no comparative study has been conducted on the importance of VLDL uptake when animals are fed soybean protein or casein without cholesterol.

Dietary protein level has also been shown to influence lipid metabolism. In rats, protein malnutrition (2 versus 20 g/100 g casein diets) depresses plasma VLDL levels and is associated with an accumulation of lipids in the liver. The reduced hepatic apolipoprotein synthesis for VLDL formation is responsible for lipid accumulation in the liver. To our knowledge no comparative study on lipoprotein metabolism in rats fed different animal or vegetable protein levels is available. Thus, whether dietary protein level affects plasma VLDL metabolism remains to be investigated.

The objective of the present study was to determine, in the absence of dietary cholesterol, the effects of dietary protein source (soybean protein and casein) and protein level (protein deficient, 10; normal, 20; or high, 30 g/ 100 g) on the liver and VLDL lipid concentrations and VLDL binding by hepatic membranes and isolated hepatocytes.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Animals and diets.

Male Wistar rats (Iffa Credo, l’Arbresle, France) weighing 80.5 g at the beginning of the experiment were housed in stainless steel cages in a room maintained at constant temperature (24°C) and humidity (60%) with a 12-h light/dark cycle. The rats were allowed free access to an adequate purified diet (20 g/ 100 g casein and 5 g/100 g olive oil) for 10 d. After this adaptation period, the rats were randomly divided into 6 groups of 10 (6 rats for blood and liver membrane preparation and 4 for hepatocytes isolation). Rats were fed diets containing 10, 20 or 30 g/ 100 g casein [95% purity (10C, 20C, 30C)] or purified soybean protein [98% purity (10S, 20S, 30S)] (Table 1). Purified soybean protein was prepared as previously described (13). Food and water were freely available. The general guidelines for the care and use of laboratory animals was followed (14). The donors for the preparation of ³H-VLDL and total lipoproteins were male Wistar rats (200–250 g) fed a standard cholesterol-free diet (UAR; Villemoisson, Epinay/orge, France).

After the 28-d diet period, rats were fasted overnight and 6 of each group were anesthetized with sodium pentobarbital (60 mg/kg body weight) prior to blood collection. Blood was collected by abdominal aorta puncture into tubes containing EDTA (final concentration 1 g/L). Plasma was prepared by low speed centrifugation (600 x g for 10 min) and preserved with 0.26 mmol/L disodium EDTA + 3 mmol/L sodium azide + 2 mmol/L butylated hydroxy toluene. The livers were removed, washed with cold saline (150 mmol/L NaCl), quickly excised, blotted and weighed. Approximately 1 g per liver was removed and homogenized in an ultraturax homogenizer (Bioblock Scientific, Illkrich, France) for lipid extraction. Approximately 100 mg of the same lobe was homogenized in a potter Elvejhem homogenizer and used for protein determination.

In vivo preparation of [³H]-VLDL.

The preparation of [³H]-VLDL was performed using the technique of Meghelli-Bouchenak et al. (15). [³H-3–4-5]-L-leucine (CEA, Gif sur Yvette, France) 3.7 GBq/mmol was diluted with pure unlabeled L-leucine (Merck, Darmstadt, Germany) in 150 mmol/L NaCl to obtain a specific radioactivity of 2.66 GBq/mmol. Ten donor rats were injected via the tail vein with 0.5 mL of this solution after being fasted for 6 h. Blood was collected into tubes containing EDTA, centrifuged at 600 x g for 10 min, and plasmas were then pooled. The density of 50-mL pooled plasma was adjusted to d = 1006 g/L with KBr (0.322 g/L plasma). After centrifugation at 270,000 x g at 15°C for 5 h in a Beckman L5–65 (65 Ti rotor; Beckman Instruments, Palo Alto, CA), floating VLDL were isolated and washed by two further centrifugations. A fraction of [³H]-leucine-VLDL was delipidated and the apolipoproteins separated by electrophoresis using SDS-PAGE (2.5–20%) to evaluate the distribution of radioactivity. The distribution of [³H]-leucine among the various VLDL-apolipoproteins was: apo B-100, 35%; apo B-48, 37%; apo E, 19%; apo A-I, 7%; and apo C, <1%. [³H]-VLDL contained: proteins, 8.8%; triglyceride, 66.2%; pyridoxal, 21.8%; free cholesterol, 10.7%; cholesteryl esters, 3.2%.

Total lipoprotein and VLDL isolation.

The density of pooled plasma from 10 donor rats was adjusted to 1.21 kg/L by the addition of cristalline KBr. Lipoproteins of density < 1.21 kg/L were separated by a single ultracentrifugation (Model L8–55 ultracentrifuge, 50 Ti rotor; Beckman) at 122,249 x g at 15°C for 48 h. Total lipoproteins were dialyzed twice against 150 mmol/L NaCl + 0.04% Na EDTA, pH 7.4 at 4°C, in spectra/Por 2 dialysis tubing (Spectrum Medical Industries, Los Angeles, CA) for 24 h. Isolation of VLDL from 2 mL plasma was carried out, and VLDL fractions (d < 1.006) were washed and dialyzed as described above.

Preparation and incubation of liver membranes.

Liver membranes were prepared according to Kovanen et al. (16) from 1 g of each liver from the largest lobe. Each fresh plasma membrane preparation was tested for 5'-nucleotidase activity (Sigma 5'-ND, procedure UV) to determine the purity of plasma membrane preparations. The values were estimated to range from 0.085 to 0.094 U/mg membrane protein and were not different among groups. Contamination with subcellular organellar membranes was not assessed; however, we was assumed that the plasma membrane preparations were of equal purity. Future studies should more carefully consider the relative enrichment of membrane fractions. The standard binding assays were carried out in triplicate in buffer C (25 mmol/L NaCl, 0.5 mmol/L CaCl₂, 50 mmol/L Tris HCl, bovine serum albumin 20 g/L, pH 8), in the presence of 80–100 µg membrane protein and 0.1, 0.2, 0.3, 0.4, 0.5 and 1 mg/L of labeled [³H]-VLDL, in the presence or absence of unlabeled lipoprotein excess (2 g/L), with a final volume of 150 µL.

Preparation and incubations of isolated hepatocytes.

Isolated hepatocytes were prepared from four rats in each dietary group, according to the method of Seglen et al. (17) modified by Skrede et al. (18). The liver was perfused in situ through veina porta for 5 min at a rate of 50 mL/min with perfusion buffer, pH 7.4 (8.3 g NaCl, 0.5 g KCl, 2.4 g HEPES, 5.5 mL mol/L NaOH and double-distilled water addition to 1 L). While still being perfused, the liver was cut from the carcass and the perfusion was then switched over to O₂-saturated collagenase buffer, pH 7.6 (3.9 g NaCl, 0.5 g KCl, 0.7 g CaCl₂ 2H₂O, 24.0 g HEPES, 0.5 g collagenase Sigma type A, 66 mL mol/L NaOH and double-distilled water addition to 1 L). After the 10-min perfusion, the liver was transferred to a petri dish and gently dispersed in Krebs-Henseleit solution with a stainless steel comb. The suspension was then purified by a succession of filtrations and centrifugations to remove nonparenchymal cells, damaged cells, subcellular debris and small clumps of nonperfused tissue (17). The cells were then resuspended in Krebs-Henseleit buffer containing 10 g/L bovine serum albumin (fatty acid free) as described previously (18). The counting and viability of cells (measured by exclusion of trypan blue) performed on a hemocytometer revealed that 90–95% of the cells were viable. For binding assay, isolated hepatocytes were incubated as described for hepatic membrane. The binding of ³H-VLDL was performed for 30 min at 37°C under constant stirring.

After incubation of hepatic membranes and isolated hepatocytes, aliquots were poured over presoaked glass fiber (Whatman GF/C) under vacuum. The filters were rapidly rinsed with 25 mL cold buffer (buffer C, and Krebs-Henseleit solution, for hepatic membrane and isolated hepatocytes, respectively), dried and counted.

Chemical analysis.

The protein contents of VLDL, hepatocytes, hepatic membrane and [³H]-VLDL were estimated according to Shacterle and Pollack (19) using bovine serum albumin as the standard. The specific radioactivity of [³H]-VLDL was measured in a liquid scintillation analyzer (1900 TR; Packard, Canbera, Meriden, CT) and estimated to 500 Bq/mg protein. VLDL apo B-100 was determined as previously described (15). The total lipids of liver were extracted according to the method of Folch et al. (20). Liver and VLDL phospholipids were estimated by phosphorus content according to the method of Bartlett (21). Plasma, VLDL and liver were assayed for triglyceride and total cholesterol with Boehringer PAP enzyme kits (Boehringer Manheim, Meylan, France) using glycerol and cholesterol as standards, respectively.

Statistical analysis.

Data are means ± SEM, n = 4 or 6. The 20 g/ 100 g protein diets were used as the reference to evaluate the effects of dietary protein levels, regardless of the protein source. When the effects of the protein source were determined, only the 20S group was compared with the 20C group. The statistical analyses of the data were performed with Statistica (version 4.1; Statsoft, 1994, Paris,France). Data were tested by two-way ANOVA followed by Fisher’s least significant difference test. Differences in the means were considered significant at P < 0.05.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Hepatic lipid concentrations

    Dietary protein source. The liver weights and hepatic phospholipid concentrations were lower but triglyceride and total cholesterol concentrations were higher in the 20S than in the 20C group (Table 2).

Plasma and VLDL lipid and apolipoprotein concentrations

    Dietary protein source. Plasma total cholesterol concentrations were not modified by the protein source, but plasma and VLDL triglyceride concentrations were lower in the 20S than in the 20C group (Table 3). The 20S group had a lower VLDL mass indicated by low phospholipid and protein contents. VLDL apo B-100 (the major VLDL apolipoprotein) contents were also lower in the soybean protein groups.

Characteristics of hepatic membranes and binding of ³H-VLDL to hepatocytes and liver membrane preparations

Protein and phospholipid concentrations in hepatic membranes were expressed per g liver (Table 4). Hepatic membrane proteins and phospholipids did not differ among the groups. A similar dissociation constant (kd) was obtained in the various incubations with hepatocytes or hepatic membranes.

    Dietary protein level. The 30C and 30S groups had lower ³H-VLDL binding to hepatocytes and liver membranes than the 20C and 20S groups. The 10S group had lower ³H-VLDL binding to liver hepatocytes than the 20S group, whereas VLDL binding to hepatocytes and the hepatic membrane did not differ between the 10C and 20C groups.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The first objective of this study was to determine whether dietary protein source affects VLDL composition and binding to the liver. Total plasma cholesterol concentrations did not differ in groups fed soybean protein and casein. The greatest effect was in triglyceride concentrations, which were lower in the plasma of rats fed soybean protein than in those fed casein. These results are in agreement with those of Horigome and Cho (22) who have shown that soybean protein compared with casein results in lower plasma triglyceride levels. The present study also demonstrates that the soybean protein diet compared with the casein diet lowered plasma VLDL particle number measured by diminished VLDL-triglyceride and -apo B-100 concentrations. Similar findings were reported in rats and mice (23) and in soy-treated postmenopausal women (24). This might, in part, result from diminished VLDL production, but Cohn et al. (9) reported that the daily production of VLDL-apo B is similar in rats fed soybean protein or casein. We have assayed VLDL uptake by liver and have provided evidence that rats fed soybean protein had a higher isolated hepatocyte uptake of VLDL than those fed casein. This enhanced uptake of VLDL by the hepatocytes of soybean protein-fed rats was supported by the data obtained with hepatocyte membranes. Therefore, the lower plasma VLDL particle number and plasma triglyceride concentrations in rats fed soybean protein compared with casein resulted from an enhanced VLDL uptake in the liver. Our results agree with those of Sirtori et al. (10) who have shown that the specific binding of VLDL to liver membranes in cholesterol-fed rats was low in those fed the cholesterol-enriched casein diet compared with those fed the cholesterol-enriched soybean protein diet. Sirtori et al. (10) suggested that the liver cholesterol enrichment induced by the casein diet reduces the hepatic high affinity receptors for VLDL. In fact, excess cholesterol suppresses cholesterol biosynthesis and downregulates hepatic B/E receptors (11). In this report, we used diets free of cholesterol and demonstrated that the casein-fed rats exhibited lowered VLDL binding to isolated hepatocytes and hepatocyte membranes than the soybean protein-fed group. This was associated, however, with lower hepatic cholesterol levels in the former group than in the latter group. These results suggest that factors other than hepatic cholesterol content may inhibit the regulation of lipoprotein binding. Our purified soybean protein may have still contained isoflavones because their removal requires an aqueous alcohol extraction process (25). Soybean isoflavones (daidzein and genistein) and equol (the main end product of bacterial degradation of daidzein) are structurally similar to estrogens and have been shown to interact with estrogen receptors and modulate lipid metabolism (26–29). Ni et al. (25) showed that the isoflavone-intact soybean protein, but not the alcohol-extracted soybean protein enhanced hepatic LDL receptor mRNA in exogenously hypercholesterolemic rats. On the other hand, the amino acid composition of proteins may account for the effects on VLDL binding. Soybean protein contains greater arginine levels than casein. Feeding soybean protein might raise plasma arginine level, and subsequently the glucagon level (30). Glucagon increases the number of hepatic LDL receptors (31).

Another aim of this study was to determine whether the dietary protein level affects VLDL composition and binding to hepatic membranes and isolated hepatocytes. Rats were fed variuos dietary protein levels (10, 20 or 30 g/100 g) to modify the dietary supply of amino acids. Our study shows that despite constant plasma total cholesterol concentrations, VLDL particle number as measured by mass and apo B-100 levels was raised with increased casein or soybean protein levels in the diet. This could result from increased VLDL production by the liver. Filho et al. (32) reported increased plasma insulin concentrations with increased casein level (6, 21 and 35%) in the diet. The role of insulin as stimulator of hepatic lipogenesis and VLDL production has been established (33). Furthermore, the enhanced number of circulating VLDL particles in rats fed high protein diets could also result from lowered VLDL catabolism. Indeed, rats fed 30 g/100 g protein diets had decreased VLDL binding to hepatic membranes and isolated hepatocytes (Table 4), indicating a reduced number and/or activity of VLDL receptors. This is supported by Divino Filho et al. (33) findings that feeding high casein diets (35 versus 21 g/ 100 g) to rats decreases plasma and erythrocyte threonine concentrations. Threonine is an essential amino acid involved in VLDL receptor biosynthesis (34).

In conclusion, in the absence of dietary cholesterol, dietary protein seems to play only a minor role in the regulation of plasma total cholesterol concentration. However, dietary protein source influences the VLDL clearance rate. Soybean protein, compared with casein, increases VLDL uptake by hepatocytes and decreases plasma triglyceride concentrations. Further research is required to determine the mechanism by which soybean protein accelerates the VLDL uptake by liver.

	ACKNOWLEDGMENTS

 
The authors thank Anne Magnet, an English-for-specific-purposes Linguist at the University of Burgundy (France), for editing the manuscript.

	FOOTNOTES

 
¹ This work was supported by the French Foreign Office with International Research Extension Grant 95MDU 318 and the Regional Council of Burgundy.

Manuscript received 18 June 2003. Initial review completed 14 July 2003. Revision accepted 2 October 2003.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

1. Carroll, K. K. (1982) Hypercholesterolemia and atherosclerosis effects of dietary protein. Fed. Proc. 41:2792-2796.[Medline]

2. Widhalm, K., Brazda, G., Schneider, B. & Kohl, S. (1993) Effect of soy protein diet versus standard low fat, low cholesterol diet on lipid and lipoprotein levels in children with familial or polygenic hypercholesterolemia. J. Pediatr. 123:30-34.[Medline]

3. Wolfe, B. M., Giovannetti, P. M., Cheng, D.C.H., Roberts, D.C.K. & Carroll, K. K. (1981) Hypolipidemic effect of substituting soybean protein isolate for all meat and dairy protein in the diet of hypercholesterolemic men. Nutr. Rep. Int. 24:1187-1196.

4. Meinertz, H., Nilausen, K. & Faergeman, O. (1990) Effects of dietary proteins on plasma lipoprotein levels in normal subjects: Interaction with dietary cholesterol. J. Nutr. Sci. Vitaminol. 36:S157-S164.

5. Teede, H. J., Dalais, F. S., Kotsopoulos, D., Liang, Y. L., Davis, S. & McGrath, B. P. (2001) Dietary soy has both beneficial and potentially adverse cardiovascular effects: a placebo-controlled study in men and postmenopausal women. J. Clin. Endocrinol. Metab. 86:3053-3060.[Abstract/Free Full Text]

6. Eklund, A. & Sjöblom, L. (1986) Effect of dietary proteins on hepatic and plasma lipids, and some properties of major plasma lipoprotein fractions during dietary-induced hypercholesterolemia in male rats. Biochim. Biophys. Acta. 877:127-134.[Medline]

7. Eisenberg, S. (1983) Lipoprotein and lipoprotein metabolism.A dynamic evaluation of the plasma fat transport system. Klin. Wochenschr. 61:119-132.[Medline]

8. Démonty, I., Deshaies, Y. & Jacques, H. (1998) Dietary proteins modulate the effects of fish oil on triglyceridemia in the rat. Lipids 33:913-921.[Medline]

9. Cohn, J. S., Kimpton, W. G. & Nestel, P. J. (1984) The effect of dietary casein and soy protein on cholesterol and very low density lipoprotein in the rat. Atherosclerosis 52:219-231.[Medline]

10. Sirtori, C. R., Galli, G., Lovati, M. R., Carrara, P., Bosisio, E. & Kienle, M. G. (1984) Effects of dietary proteins on the regulation of liver lipoprotein receptors in rats. J. Nutr. 114:1493-1500.

11. Roach, P. D., Balasubramaniam, S., Hirta, F., Abbey, M., Szanto, A., Simons, L. A. & Nestel, P. J. (1993) The low-density lipoprotein receptor and cholesterol synthesis are affected differently by dietary cholesterol in rat. Biochim. Biophys. Acta 1170:165-172.[Medline]

12. Sultan, F., Benhizia, F., Lagrange, D., Will, H. & Griglio, S. (1995) Effects of dietary cholesterol on activity and mRNA levels of hepatic lipase in rat. Life Sci 56:31-37.[Medline]

13. Madani, S., Lopez, S., Blond, J. P., Prost, J. & Belleville, J. (1998) Highly purified soybean protein is not hypocholesterolemic in rats but stimulates cholesterol synthesis and excretion and reduces polyunsaturated fatty acid biosynthesis. J. Nutr. 128:1084-1091.[Abstract/Free Full Text]

14. Council of European Communities (1986) Council instructions about the protection of living animals used in scientific investigations. Off. J. Euro. Comm. (JO. 86/609/CEE) L358:1-28.

15. Meghelli-Bouchenak, M., Belleville, J., Boquillon, M., Scherrer, B. & Prost, J. (1991) Removal rates of rat serum [³H] labeled very low density lipoproteins after consumption of two different low protein diets. Nutr. Res. 11:587-597.

16. Kovanen, P. T., Brown, M. S. & Goldstein, J. L. (1979) Increased binding of low density lipoprotein to liver membrane from rats treated with 17-ethyl oestradiol. J. Biol. Chem. 254:11367-11373.[Free Full Text]

17. Seglen, P. O. (1973) Preparation of rat liver cells: Enzymatic Requirements for Tissue Dispersion. Exp. Cell Res. 82:391-398.[Medline]

18. Skrede, S., Narce, M., Bergseth, E. & Bremer, J. (1989) The effect of alkylthioacetic acids (3-thia fatty acids) on fatty acid metabolism in isolated hepatocytes. Biochim. Biophys. Acta 1005:296-302.[Medline]

19. Schacterle, G. R. & Pollack, R. L. (1973) Simplified method for the quantitative assay of small amounts of proteins in biological materials. Anal. Biochem. 51:654-655.[Medline]

20. Folch, J., Lees, M. & Sloane Stanley, G. H. (1957) A simple method for isolation and purification of total lipids from animal tissues. J. Biol. Chem. 226:497-509.[Free Full Text]

21. Bartlett, G. R. (1958) Phosphorus assay in column chromatography. J. Biol. Chem. 234:466-468.

22. Horigome, T. & Cho, Y. S. (1992) Dietary casein and soybean protein affect the concentrations of serum cholesterol, triglyceride and free amino acids in rats. J. Nutr. 122:2273-2282.

23. Nagata, Y., Tanaka, K. & Sugano, M. J. (1981) Serum and liver cholesterol levels of rats and mice fed soy-bean protein or casein. Nutr. Sci. Vitaminol. (Tokyo) 27:583-593.

24. Vigna, G. B., Pansini, F., Bonaccorsi, G., Albertazzi, P., Donega, P., Zanotti, L., De Aloysio, D., Mollica, G. & Fellin, R. (2000) Plasma lipoproteins in soy-treated postmenopausal women: a double-blind, placebo-controlled trial. Nutr. Metab. Cardiovasc. Dis. 10:315-322.[Medline]

25. Ni, W., Yoshida, S., Tsuda, Y., Nagao, K., Sato, M. & Imaizumi, K. (1999) Ethanol-extracted soy protein isolate results in elevation of serum cholesterol in exogenously hypercholesterolemic rats. Lipids 34:713-716.[Medline]

26. Xu, X., Duncan, A. M., Merz, B. E. & Kurzer, M. S. (1998) Effects of soy isoflavones on oestrogen and phytoestrogen metabolism in premenopausal women. Cancer Epidemiol. Biomarkers Prev. 7:1101-1108.[Abstract/Free Full Text]

27. Greaves, K. A., Wilson, M. D., Rudel, L. L., Williams, J. K. & Wagner, J. D. (2000) Consumption of soy protein reduces cholesterol absorption compared to casein protein alone or supplemented with an isoflavones extract or conjugated equine estrogen in ovariectomized cynomolgus monkeys. J. Nutr. 130:820-826.[Abstract/Free Full Text]

28. Parini, P., Angelin, B., Stavreus-Evers, A., Freyschuss, B., Eriksson, H. & Rudling, M. (2000) Biphasic effects of the natural estrogen 17 beta-estradiol on hepatic cholesterol metabolism in intact female rats. Arterioscler. Thromb. Vasc. Biol. 20:1817-1823.[Abstract/Free Full Text]

29. Hwang, J., Wang, J., Morazzoni, P., Hodis, H. N. & Sevanian, A. (2003) The phytoestrogen equol increases nitric oxide availability by inhibiting superoxide production: an antioxidant mechanism for cell-mediated LDL modification. Free Radic. Biol. Med. 34:1271-1282.[Medline]

30. Sugano, M., Ishiwaki, I. & Nakashima, K. (1984) Dietary protein-dependent modification of serum cholesterol levels in rats: significance of the arginine to lysine ratio. Ann. Nutr. Metab. 28:192-199.[Medline]

31. Rudling, M. & Angelin, B. J. (1993) Stimulation of rat hepatic low density lipoprotein receptors by glucagon.Evidence of a novel regulatory mechanism in vivo. Clin. Invest. 91:2796-2805.

32. Divino filho, J. C., Hazel, S. J., Anderstam, B., Bergström, J., Lewitt, M. & Hall, K. (1999) Effect of protein intake on plasma and erythrocyte free amino acids and serum IGF-I and IGFBP-1 levels in rats. Endocrinol. Metab. 40:E693-E701.

33. Sparks, J. D. & Sparks, C. E. (1994) Insulin regulation of triacylglycerol-rich lipoprotein synthesis and secretion. Biochim. Biophys. Acta 1215:9-32.[Medline]

34. Iijima, H., Miyazawa, M., Sakai, J., Magoori, K., Ito, M. R., Suzuki, H., Nose, M., Kawarabayasi, Y. & Yamamoto, T.T.J. (1998) Expression and characterization of a very low density lipoprotein receptor variant lacking the O-linked sugar region generated by alternative splicing. J. Biochem. 124:747-755.[Abstract/Free Full Text]

This article has been cited by other articles:

				N. L. Escudero, F. Zirulnik, N. N. Gomez, S. I. Mucciarelli, and M. S. Gimenez Influence of a Protein Concentrate from Amaranthus cruentus Seeds on Lipid Metabolism Experimental Biology and Medicine, January 1, 2006; 231(1): 50 - 59. [Abstract] [Full Text] [PDF]

				M. Lacroix, C. Gaudichon, A. Martin, C. Morens, V. Mathe, D. Tome, and J.-F. Huneau A long-term high-protein diet markedly reduces adipose tissue without major side effects in Wistar male rats Am J Physiol Regulatory Integrative Comp Physiol, October 1, 2004; 287(4): R934 - R942. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Madani, S. Articles by Belleville, J. Search for Related Content PubMed PubMed Citation Articles by Madani, S. Articles by Belleville, J.