Casein-induced hypercholesterolemia in rabbits: distribution of cholesterol, triglycerides and phospholipids between serum and liver

Replacement of soy protein by casein in the cholesterol-free, semipurified diet of rabbits caused hypercholesterolemia within 7 days. After 36 days, the serum of casein-fed rabbits displayed elevated levels of free and esterified cholesterol and phospholipids, but not of triglycerides. Most of the excess of serum cholesterol in the casein group was localized in the LDL fraction, but there were marked variations in the density profile of the serum lipoproteins between individual rabbits. Dietary casein induced an increased content in liver of free and esterified cholesterol, but not of phospholipids and triglycerides. The molar ratio of free to esterified cholesterol in the liver was decreased by casein. In contrast, feeding casein resulted in an increase of this ratio in the serum.     

Effects of casein and soy protein on hepatic and serum lipids and lipoprotein lipid distributions in the rat

Rats fed a semipurified diet containing casein developed higher levels of circulating triglycerides and cholesterol than animals fed a soy protein-containing diet. The increased serum lipid levels in non-fasted rats were associated largely with the d less than 1.006 g/ml lipoprotein particles (e.g. chylomicrons or very low density-like lipoproteins). In addition, casein-fed rats exhibited higher levels of circulating insulin and depressed hepatic 7 alpha-hydroxylase levels compared to soy-fed rats. Supplementation of the casein diet with arginine, to give an arginine/lysine ratio comparable to that in the soy diet, resulted in a reduction of d less than 1.006 g/ml lipids, a reduction in serum insulin levels and an elevation in hepatic 7 alpha-hydroxylase activity. Supplementation of the soy diet with lysine also resulted in modification of these parameters toward those observed with casein diets, albeit the effects were less dramatic. The results suggest that the hyperlipidemia associated with feeding casein-based diet is associated with decreased rates of clearance of chylomicron-like lipoproteins and their component triglycerides and cholesterol. Furthermore, this is largely prevented by addition of arginine to diets containing casein as the sole protein source.     

Effects of soy protein and saponins on serum, tissue and feces steroids in rat

Four groups of rats were fed, for 45 days, one of the following semipurified diets containing sucrose 55% (w/w) and (a) casein 25%, (b) casein 24%, saponins (from Saponaria officinalis) 1%, (c) isolated soy protein 25%, (d) soy protein 24%, saponins 1%. The soy protein diet, compared to the casein one, produced an increase in the fecal excretion of neutral sterols on the 29th and 42nd days, without any modification in the liver, aorta and serum cholesterol concentrations. The effect of soy protein cannot be attributed to its saponin content but other substances associated to soy protein may interfere. With the casein diet, added saponins increased the fecal excretion of neutral sterols and bile acids and decreased liver and aorta cholesterol levels. Serum cholesterol was found unchanged. The effects of saponins were suppressed or greatly reduced with the soy protein diet. These results could be explained by binding of the sterols in insoluble forms.     

Influence of fish protein as compared to casein and soy protein on serum and liver lipids, and serum lipoprotein cholesterol levels in the rabbit

Serum and hepatic cholesterol and triglyceride levels, and serum lipoprotein cholesterol were investigated in rabbits fed fish protein as compared to casein and soy protein as part of a 20% protein, low fat, cholesterol-free, semi-purified diet. A nonpurified diet was used as a control. After a 28-day experimental period, rabbits fed casein developed hypercholesterolemia compared to those fed the soy protein diet. Serum cholesterol levels of rabbits fed fish protein was intermediate and not different from that of the casein or the soy protein group. However, serum triglycerides were higher in the fish group than in the casein group. Feeding of fish protein resulted in a reduction of hepatic cholesterol compared to casein, indicating no direct relationship between serum and hepatic cholesterol. In addition, fish protein induced a decrease of cholesterol in the low density lipoproteins (LDL) compared to casein and an increase of cholesterol in the high density lipoproteins (HDL) compared to casein and soy protein. Reduction in LDL-cholesterol (LDL-C) and elevation in HDL-cholesterol (HDL-C) caused a 10-fold decrease in the LDL-C/HDL-C ratio of fish protein fed rabbits compared to those fed casein. This ratio was similar to that observed with soy protein which was also lower than the ratio of the casein group. Thus, since the LDL-C/HDL-C ratio has been shown to be a good indicator of the atherosclerosis risk, these results suggest that fish protein, as well as soy protein, may reduce the risk of atherosclerosis in rabbits, compared to casein.     

Effects of cholesterol-free, semipurified diets containing different levels of casein or soy protein on distribution of cholesterol and protein among serum lipoproteins of rabbits

Rabbits fed cholesterol-free, low-fat, semipurified diets have more cholesterol and protein in serum low density lipoprotein (LDL) relative to high density lipoprotein (HDL) than rabbits fed Chow diet. This difference was accentuated by a casein semipurified diet but was also observed with a soy protein diet even though the latter did not produce an elevation of serum cholesterol. To investigate the reason for these differences, the formulation of the semipurified diets was altered by reducing the level of protein from 27 to 16%, increasing the fat from 1 to 4% and the fiber from 5 to 13%, to correspond more closely to the proportions in Chow. With this formulation, the soy protein diet gave a lipoprotein pattern similar to that of Chow, whereas the casein diet produced a moderately elevated serum cholesterol level with more cholesterol in LDL than in HDL. When the protein in the newly formulated diets was increased back to 27%, the lipoprotein patterns reverted to those obtained with the original formula. In this case, soy protein-fed rabbits had moderately elevated serum cholesterol whereas casein-fed animals showed hypercholesterolemia. These results indicate that the altered lipoprotein pattern observed previously in rabbits fed semipurified diets is related to the high level of protein in those diets.     

Compositional changes in serum lipoproteins during developing hypercholesterolemia induced in rabbits by cholesterol-free, semipurified diets

Changes in the concentration and composition of serum very low density lipoprotein (VLDL), low density lipoprotein (LDL) and high density lipoprotein (HDL) were studied in rabbits transferred from chow diet to cholesterol-free, semipurified diets containing casein or isolated soy protein. The fat and fibre content of these diets was similar to that of chow but a higher protein level was used to enhance the hypercholesterolemia. During the first week on the casein diet, there was a marked increased in LDL-cholesterol, protein and phospholipids, and these higher levels were maintained during the subsequent 3 weeks of the study. Similar but less marked changes were obtained with the soy protein diet. The components of VLDL showed relatively little change after introduction of the diets to the animals. In both VLDL and LDL, the proportion of cholesterol increased and that of triglycerides decreased after 1 week on the casein diet and a similar trend was seen in HDL. The concentration of HDL-cholesterol showed little change but triglycerides, protein and phospholipids all tended to decline on both casein and soy protein diets.     

The effect of dietary casein and soy protein on cholesterol and very low density lipoprotein metabolism in the rat

Rats fed a high-cholesterol semipurified diet containing casein developed higher levels of serum cholesterol than soy-fed animals. The hypercholesterolaemia of casein-fed rats was due to accumulation of very low density lipoproteins (VLDL), as measured by increased concentrations of serum VLDL cholesterol, protein and apoprotein B. High density lipoprotein (HDL) cholesterol was similar for the two dietary groups. Cholesterol absorption, as measured by the dual isotope ratio method and by direct measurement of cholesterol secretion into thoracic duct lymph, did not differ between the two groups. Cholesterol kinetics were derived from plasma cholesterol specific radioactivity curves and the casein-fed rats had a similar rate of plasma cholesterol production, but a significantly lower plasma cholesterol fractional catabolic rate (FCR) compared with the soy-fed rats. Kinetics of plasma VLDL apoprotein B, derived from analysis of reinjected 125I-labelled VLDL protein, also showed a lower fractional catabolic rate with casein feeding. This suggests that the accumulation of VLDL in the plasma of rats fed dietary casein is not due to excess VLDL production but to deficient VLDL removal. The hypercholesterolaemia appears to be a consequence of diminished VLDL catabolism.     

Intermediate density lipoprotein-apolipoprotein B turnover in rabbits fed semipurified diets containing casein or soy protein

The aim of this study was to investigate the underlying causes of elevated levels of intermediate density lipoprotein (IDL) in rabbits fed casein compared to soy protein in a cholesterol-free, semipurified diet. Kinetic studies were carried out in which homologous, radiolabelled IDL was injected into casein- and soy protein-fed animals after a period of 7 months on diet. Total plasma cholesterol and IDL cholesterol and protein were significantly higher in animals fed casein compared to those fed soy protein. The fractional catabolic rate of IDL-apolipoprotein B was significantly lower in casein-fed animals but the difference in mean values for the production rate did not reach statistical significance. The effect of feeding casein on IDL can be attributed to its reduced efficiency of removal from the plasma.     

Relationship between dietary proteins, their in vitro digestion products, and serum cholesterol in rats

A relationship was assessed between the amino acid composition of 9 protein sources or of their in vitro digestion products and total serum cholesterol in rats. Three animal proteins (casein, beef, fish) and 6 vegetable proteins (soy, pea, peanut meal, rapeseed, oatmeal, wheat gluten) were tested. The intact protein sources were submitted to an enzymatic proteolysis according to a new in vitro digestion method. Each protein source was hydrolyzed for 30 min with pepsin at pH 1.9, then with 10 mg pancreatin at basic pH in a dialysis cell. The digestion products diffused through the dialysis membrane of the cell and were collected by a circulating sodium phosphate buffer over a 6-h period. They were likely to correspond to end products luminal in vivo digestion. The aromatic and the basic amino acids were present in higher proportions in the digestion products than in the intact protein sources, reflecting the specificity of the proteolytic enzymes. Total serum cholesterol was measured on male Sprague-Dawley rats fed cholesterol-free or cholesterol-enriched (1% cholesterol, 0.5% cholic acid) semipurified diets containing protein sources. Total serum cholesterol ranged from 70 mg/dl with the pea diet to 98 mg/dl with the peanut meal diet in rats fed cholesterol-free diets and from 163 mg/dl with the wheat gluten diet to 313 mg/dl with the casein diet in rats fed the cholesterol-enriched diets. These results suggested no specific effect of protein from animal or vegetable origin on total serum cholesterol in rats. In rats fed cholesterol-enriched diets, significant correlations were observed between total serum cholesterol and tyrosine content or leucine/isoleucine ratio of digestion products. These correlations were stronger than those observed with intact protein sources.     

Effects of soy components on blood and liver lipids in rats fed high-cholesterol diets

AIM: To assess the effects of soy protein, isoflavone, and saponin on liver and blood lipid in rats that consumed high-cholesterol diets. METHODS: High-cholesterol diets (1%) with or without soy material were fed to 6-wk-old male Sprague-Dawley rats for 8 wk. Blood lipids, liver lipids, glutamic oxaloacetic transaminase (GOT), and glutamic pyruvic transaminase (GPT) levels were measured. The in vitro bile acid-binding ability of soy materials was analyzed. RESULTS: The results of in vitro studies showed that soy protein isolate had a significantly higher bile acid-binding ability (8.4±0.8%) than soy saponin (3.1±0.7%) and isoflavone (1.3±0.4%, P<0.05). On the other hand, at the end of the experimental period, rats that consumed soy protein diets had lower GOT and GPT levels than rats that consumed casein under high-cholesterol diets. Rats that consumed soy protein also had lower total cholesterol (TC) levels in the liver than those that consumed casein under high-cholesterol diets. Rats that consumed the soy protein diet containing both saponin and isoflavone had lower hepatic TC level than those that consumed the soy protein diet without isoflavone alone. The effect of different types of proteins on triglyceride was not significant. CONCLUSION: Consumption of soy provided benefits to control lipid levels under high-cholesterol dieting conditions in this rat model of hypercholesterolemia. The major component that reduced hepatic TC was not saponin, but possibly isoflavone.     

Lipoprotein and apolipoprotein distribution in Zucker rats following an endurance running program

We examined the response to 12 weeks of endurance running of the obese Zucker rat and its lean littermate with regard to changes in serum lipids, lipoproteins and apoproteins. The obese Zucker rat is hyperlipoproteinemic, characterized by elevated serum triglyceride and cholesterol levels primarily associated with the very low density lipoprotein (VLDL) fraction. In lean Zucker rats, training did not affect the concentrations of serum lipids or apolipoproteins. In marked contrast, obese Zucker rats that were trained had significant decreases in serum concentrations of triglycerides, free and esterified cholesterol, and apolipoprotein B compared to their sedentary counterparts. Training obese rats caused an increase in the serum concentration of apolipoprotein E (HDL fraction). In contrast, training did not affect the concentration of Apo E in lean rats. The VLDL fraction was most affected by training obese rats showing marked 50-65% decreases in VLDL triglyceride and VLDL cholesterol. HDL cholesterol was unchanged in lean rats whereas training prompted a 29% increase in obese rats. These data show that exercise training altered the metabolic abnormalities of obese Zucker rats which are responsible for the accumulation in serum of VLDL, lipids and apolipoproteins.     

Experimental atherosclerosis in rabbits fed cholesterol-free diets. 13. Interaction of proteins and fat

The atherogenic and cholesterolemic effects of animal protein vis-a-vis plant protein are well documented. Virtually all the studies were carried out using diets high in saturated fat, such as coconut oil. In order to determine if the same effects were seen with less saturated fat, we have compared atherogenic effects of an animal protein (casein) with those of a plant protein (soybean protein isolate) fed with partially hydrogenated soybean oil (PHS) (iodine value 72) or soybean oil (iodine value 134) as part of a cholesterol-free semipurified diet. After 6 months only rabbits fed casein-PHS exhibited elevated levels of plasma and liver cholesterol and triglycerides and atherosclerosis. Rabbits fed soy protein-PHS had slightly higher plasma cholesterol and triglycerides than did those fed soy protein and soybean oil, but values in both groups were in the normal range. The different effects of animal and plant protein on lipidemia and atherosclerosis can be influenced by dietary fat and appear to be dependent on fat saturation.     

Intestinal apolipoprotein B-48 synthesis and lymphatic cholesterol transport are lower in swine fed high fat, high cholesterol diet with soy protein than with casein

Effects of dietary proteins on intestinal lipoproteins were studied in 8 Yorkshire swine fed a high fat, high cholesterol diet with either casein or soy protein. After 5 weeks of feeding, the casein group exhibited moderately elevated levels of serum cholesterol (334 +/- 46 mg/dl). The soy protein group showed significantly less hypercholesterolemia as compared to the casein group (122 +/- 8 mg/dl). Swine were subjected to cannulation of mesenteric lymph duct under halothane anesthesia. A single dose of 250 microCi [14C]cholesterol and 10 mCi [3H]leucine was infused into the upper jejunum 2 h after one-fifth of daily food was given. The 3-h lymphatic transport of cholesterol in casein-fed swine was significantly higher than in those fed soy protein. Triglyceride transport values were similar in the 2 groups. The [3H]leucine incorporation study revealed that transport of apo B-48 bore a significant positive relationship to transport of cholesterol in both chylomicron and VLDL fractions of mesenteric lymph. A greater apo B-48 secretion with higher specific activity was probably responsible for the greater transport of cholesterol in chylomicrons in casein-fed than in soy protein-fed swine. Similarly, the transport of lymph VLDL cholesterol in swine fed casein or soy protein paralleled the amount of accompanying apo B-48. Dietary proteins probably influence the intestinal synthesis of apo B-48 which in turn affects cholesterol transport into the lymphatics.     

Effect of alfalfa meal on shrinkage (regression) of atherosclerotic plaques during cholesterol feeding in monkeys.

A semipurified diet containing 1.2 mg of cholesterol/Cal was fed to cynomolgus monkeys (Macaca fascicularis). At the end of 6 months, a group of 18 animals was killed for evaluation of atherosclerosis in the aorta and the coronary arteries. The remaining monkeys were assigned to three groups of 18 animals each and fed, during the following 18 months, semipurified diets containing 0.34 mg of cholesterol/Cal with or without alfalfa meal, or a diet consisting entirely of Monkey Chow. a decrease in cholesterolemia and plasma phospholipid levels, normalization in the distribution of plasma lipoproteins, and reduction in the extent of aortic and coronary atherosclerosis were observed in monkeys fed the semipurified diet containing alfalfa, although the intake of cholesterol remained as high as in the usual American diet. These changes, also observed in monkeys fed a chow diet almost devoid of cholesterol, suggest that alfalfa counteracts the atherogenic effect of dietary cholesterol.     

Lipolytic activities in rats fed a sucrose-rich diet supplemented with either cystine or cholesterol: relationships with lipoprotein profiles

To study the relationships between lipolytic activities and plasma lipoprotein levels in rats, three diets were given for 8 weeks: a semipurified diet (based on sucrose, casein and lard) and this diet enriched with 5% cystine or with 1% cholesterol. Both supplemented diets induced hypercholesterolemia. Lipoprotein analysis by density gradient ultracentrifugation of plasma indicated that hypercholesterolemia of cystine-fed rats (+52%) was characterized by an increased cholesterol level in high-density lipoprotein (HDL; +131%) and low-density lipoprotein 2 (LDL2; +147%), the lipoprotein fraction containing essentially apolipoprotein-E-rich high-density lipoproteins (HDL1), and was associated with a decreased cholesterol level in triglyceride-rich lipoproteins (TRL: -69%). That obtained by cholesterol feeding (+28%) was due to a large increase in the TRL cholesterol level (+315%) whereas cholesterol was reduced in HDL (-40%) and in LDL2 (-60%). Under these dietary conditions, the activity of hepatic lipase (HL) was measured in liver homogenates and those of both HL and lipoprotein lipase were measured in plasma after heparin injection. The activity of HL (1,783 +/- 132 mU/g liver in control rats) was increased by 48% in cystine-fed rats and decreased by 40% in cholesterol-fed rats. Similar changes were observed in the activity of both lipases measured in postheparin plasma. Highly significant positive correlations linked each lipolytic activity with the level of cholesterol, phospholipids and proteins in LDL2 (HDL1-rich fraction) and in HDL. In contrast, significant negative correlations were found between all of the TRL components and the activity of the lipases.(ABSTRACT TRUNCATED AT 250 WORDS)     

Serum cholesterol, lecithin-cholesterol acyltransferase, and hepatic hydroxymethylglutaryl coenzyme A reductase activities of lean and obese Zucker rats

Serum cholesterol concentrations, lecithin-cholesterol acyltransferase (LCAT), and hepatic 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase activities of lean and obese Zucker rats were compared. The excess serum cholesterol of the female obese rat is found to be mainly free cholesterol associated with very low-density lipoproteins, whereas that of the male obese rat is carried as cholesterol esters associated with high-density lipoproteins. The high level of serum free cholesterol in the female obese rat is not due to a deficiency in lecithin-cholesterol acyltransferase activity. This enzyme activity is found to be elevated in the male obese rat. Hepatic HMG-CoA reductase activity declines as rats mature; this observation is most apparent in obese male rats. Lean rats exhibit the normal diurnal rhythm, but mature obese rats show little diurnal variation in HMG-CoA reductase activity. Obese female rats maintain high reductase activities, but the activities of obese male rats remain low at all times. Starvation suppresses liver HMG-CoA reductase and serum cholesterol in both lean and obese female rats. Thus, an increase in hepatic cholesterol synthesis may contribute to hypercholesterolemia in the obese female Zucker rat. On the other hand, factors such as nonhepatic synthesis or a decreased cholesterol catabolism may play more important roles in maintaining high serum cholesterol in the obese male Zucker rat.     

Dietary protein, fat, and minerals in nephrocalcinosis in female rats.

Young female rats fed semipurified diets containing casein or a soy protein isolate had extensive nephrocalcinosis at the junction between the outer and inner stripe of the outer medullary zone after 5 wk on the diets, whereas rats fed a diet containing a lactalbumin concentrate did not. Although the percentages of actual protein and of total ash were similar in all three diets, the concentrations of individual minerals were not, owing to methods used in isolating the proteins. Comparison of the individual mineral contents of these diets with those in other laboratories as compiled from the literature suggested that factors other than minerals, including protein, are also implicated. Dietary fat appeared to be another such factor in a series of experiments in which saturated medium-chain triglycerides and corn oil were included in diets containing soy protein isolate. Although these diets had identical mineral compositions, the rats fed medium-chain triglycerides had less severe lesions.     

Cholesterol homeostasis in lean and obese male Zucker rats

Studies were performed in male Zucker rats to determine the metabolic effect of genetic obesity on whole body cholesterol homeostasis. Lean and obese mature Zucker rats were studied during intake of either a chow diet or a semisynthetic diet containing 10% corn oil; in addition growing animals were studied during constant body weight gain on a chow diet. Under all conditions the obese Zucker rats had significantly higher levels of total plasma cholesterol and triglyceride; however, measurements of the specific activity of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase and of the rate of whole body cholesterol synthesis by sterol balance techniques demonstrated that the lean and obese animals did not differ in their endogenous rates of cholesterol synthesis. When sterol balance data were calculated per kilogram body weight, lean male Zucker rats synthesized a greater amount of cholesterol per day than obese animals. These studies demonstrate that the obese male Zucker rat, in many ways a model of human obesity, does not overproduce cholesterol and thus fails to exhibit one of major characteristics of the obese human.     

The hypocholesterolemic action of the undigested fraction of soybean protein in rats

Soybean protein was exhaustively digested with endo- and exo-type microbial proteases and the effect of the digestible low molecular fraction (LMF) and the undigested high molecular fraction (HMF) on the serum cholesterol level was compared to that of the intact protein in rats given a cholesterol-enriched diet. The HMF, peptides relatively abundant in hydrophobic amino acids, was found to be substantially hypocholesterolemic when fed at the nitrogen level equivalent to that of the 20% soybean protein diet, and not only serum but also liver cholesterol levels were similar to those usually encountered in rats given diets free of cholesterol. There was a dose-dependent reduction of serum and liver cholesterol when casein was replaced stepwise with HMF. The cholesterol-lowering action could be attributable to an increased fecal steroid excretion.