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Laboratory of Veterinary Biochemistry and * Departments of Nutrition and Laboratory Animal Science, Graduate School of Animal Health, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands
2To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • sphingomyelin • dietary casein • liver • lipoproteins • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Kritchevsky 1979
). Dietary
casein enhances cholesterol absorption and reduces fecal bile acid
secretion, leading to increased hepatic cholesterol levels. This
elicits various reactions, including an increased output of VLDL by the
liver, which results in an elevated concentration of VLDL cholesterol
(Beynen 1990
).
In previous studies with rats fed diets containing extra cholesterol
(Geelen et al. 1995
), pectin (Bladergroen et al. 1999
) or olive oil (Geelen and Beynen 1999
), we
showed that diet-induced changes in VLDL cholesterol levels were
associated with parallel changes in the amount of sphingomyelin in the
VLDL fraction. In this study, we tested whether dietary casein vs.
soybean protein would increase VLDL-sphingomyelin in rats. We have
attempted to describe how dietary casein affects hepatic sphingomyelin
metabolism.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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C12-NBD ceramide was purchased from Molecular Probes
(Eugene, OR). The origin of other chemicals has been described
previously (Geelen et al. 1995
).
Animals and diets.
Female outbred Wistar rats (HsdCpb:Wu, Harlan-CPB, Zeist, The
Netherlands), aged 3 wk, were used. They were housed in groups of three
per cage in a room with a 12-h light:dark cycle (lights on, 0700–1900 h). All rats were fed a diet containing soybean protein for 7 d.
The composition of the diet was as follows (g/100 g): soybean protein,
35; methionine, 0.3; coconut fat, 9; soybean oil, 1; cholesterol, 1;
glucose, 44.8; cellulose, 3; calcium carbonate, 1.2; monosodium
phosphate, 1.5; magnesium carbonate, 0.2; potassium chloride, 0.8;
mineral premix, 1; and vitamin premix, 1.2. Before the ingredients were
mixed, the cholesterol component of the diet was dissolved in the
coconut part of the diet. The composition of the vitamin and mineral
premixes was described by Verbeek et al. (1993)
. After
7 d (d 0 of the experiment), the rats were divided into two groups
of 18 and one group of 6, stratified for body weight. One group of 18
rats continued to receive the diet with soybean protein; the other
group of 18 rats was transferred to the same diet, but with 35.3 g
casein/100 g replacing the soybean protein and methionine. Rats had
free access to food and tap water.
Collection and preparation of samples.
Samples were taken exactly as described before (Geelen et al. 1995
)
Lipoproteins were isolated from fresh plasma by density gradient
centrifugation (Terpstra et al. 1981
). VLDL, LDL and HDL
were collected on the basis of their densities as before (Geelen et al. 1995
). Isolated lipoprotein fractions were frozen and
stored at -20°C until analysis.
Several pieces of liver were homogenized separately and used for lipid
extraction and subcellular fractionation as described previously
(Geelen et al. 1995
).
Enzyme assays.
Activities of sphingomyelinase (EC 3.1.4.12) were determined in liver
preparations. Acid sphingomyelinase activity was determined in liver
homogenates as described before (Geelen et al. 1995
).
Assays were conducted at pH 4.4 for 60 min at 37°C. Neutral
sphingomyelinase activity was determined in isolated plasma membranes.
The last-mentioned assay was performed similarly to the one for
acid sphingomyelinase except that the buffer was 50 mmol/L Tris-HCl
(pH 7.4) and the incubation was carried out in the presence of 40
mmol/L MgCl2 for 20 min.
Determination of the activity of serine palmitoyltransferase (EC
2.3.1.50) was carried out as described (Geelen et al. 1995
).
The activities of phosphatidylcholine:ceramide
cholinephosphotransferase and phosphatidylethanolamine:ceramide
ethanolaminephosphotransferase were determined essentially as described
by Vos et al. (1995)
. Briefly, the assay mixture
contained the following in a total volume of 250 µL:
26 µmol/L
12-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino-lauryl-ceramide (NBD), 174
µmol/L egg phosphatidylcholine or
phosphatidylethanolamine, 50 mmol/L Tris-HCl (pH 7.4), 5 mmol/L
EDTA, 20 µmol Triton X-100/L and 50
µL purified plasma membrane. Control experiments
demonstrated that the assays were linear with protein up to at least
200 µg of plasma membrane protein and with time, for
at least 3 h. For routine purposes, assays were conducted for 60
min with ~100 µg of plasma membrane protein. Lipids
were extracted according to Bligh and Dyer (1959)
and
separated by TLC on silica G-60 plates using chloroform/methanol/25%
ammonium hydroxide/water (70:30:4:1, v/v/v/v) as developing solvent.
NBD-ceramide spots were detected under UV-light and scraped
from the plates. For quantification, the NBD-ceramides were excited
at 465 nm and their fluorescence was measured at 530 nm. The
fluorimetry was carried out with a Perkin Elmer Luminescence
Spectrometer LS-2B.
Chemical analyses.
Cholesterol and sphingomyelin in plasma, liver homogenates and
lipoprotein fractions were isolated and quantified exactly as described
previously (Geelen et al. 1995
).
Statistical analysis.
Results shown represent the means ± SD The data within the two dietary groups for the three time points are independent so that statistical analysis of diet effects for each time point was performed by two-tailed Student’s t test. The level of significance was preset at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Plasma cholesterol was greater in the casein group than in the soybean
group on d 21. The liver cholesterol level in casein-fed rats was
significantly greater throughout the experiment (Fig. 1
). Casein feeding did not significantly influence plasma and liver
concentrations of sphingomyelin (Fig. 1)
.
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, rats fed casein had more cholesterol in the VLDL fraction on d 14 and
21. Casein consumption induced a rapid increase in
VLDL-sphingomyelin (Fig. 2B
), but diminished LDL
sphingomyelin (Fig. 2D
). The sphingomyelin concentration of
the HDL-2 particles was reduced by consumption of casein (Fig. 2F
), but the cholesterol concentration of HDL-2 was not
affected (Fig. 2E
).
|
). Casein feeding for 7 d also significantly increased the
activities of the sphingomyelin-synthesizing enzymes in hepatic
plasma membranes, phosphatidylcholine:ceramide
phosphocholinetransferase and phosphatidylethanolamine:ceramide
phosphoethanolaminetransferase (Fig. 4
). The activity of sphingomyelinase in the lysosomes was significantly
reduced after 7 d of feeding the casein diet and that in the
plasma membrane fraction was significantly raised (Fig. 5
). Again, the casein-induced differences in enzyme activities were
not seen after d 7 of the experiment.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Zhang et al. 1992
), the rats fed the
cholesterol-rich diet containing casein accumulated more
cholesterol in liver and VLDL than did their counterparts fed soybean
protein. The new finding is that consumption of casein caused a
transient increase in the amount of sphingomyelin in the VLDL fraction.
The increase on d 7 of the feeding trial was >60%. The
casein-induced increase in VLDL-sphingomyelin was associated
with decreases in the concentrations of sphingomyelin in LDL and HDL-2.
The various changes in lipoprotein-sphingomyelin, including the
transient nature of the changes, resulted in the outcome that dietary
casein did not significantly affect sphingomyelin concentrations in
whole plasma. In essence, the cholesterol and sphingomyelin
concentrations in whole plasma and lipoprotein fractions showed similar
time courses.
The amount of sphingomyelin in VLDL may reflect hepatic secretion of
this phospholipid. An increased hepatic demand for sphingomyelin to be
secreted with VLDL can be satisfied by increasing the rate of synthesis
and/or uptake from circulating lipoproteins, and/or by decreasing the
rate of catabolism. Determination of the activity of a number of key
enzymes in the synthesis of sphingomyelin indicated that on d 7 of the
experiment, the feeding of casein had increased the activities of
serine palmitoyltransferase, phosphatidylcholine:ceramide
phosphocholinetransferase and phosphatidylethanolamine:ceramide
phosphoethanolaminetransferase. On the other hand, the activity of acid
sphingomyelinase, the key enzyme in the degradation of hepatic
sphingomyelin, was significantly decreased on d 7. Taken together, the
enzyme data suggest that the extra sphingomyelin in VLDL may result
from an increase in the rate of hepatic formation and a decrease in
catabolism. This statement implies that we assume that the observed
decrease in the activity of lysosomal sphingomyelinase is more
important with respect to overall hepatic sphingomyelin catabolism than
is the observed increase in the activity of membrane sphingomyelinase.
It is difficult to see that the increased demand for hepatic
sphingomyelin in the rats fed casein is met by increased sphingomyelin
uptake. The high hepatic cholesterol concentration in these rats will
cause down-regulation of the number of LDL receptors (Brown and Goldstein 1981
), which leads to reduced LDL uptake.
The casein-induced increase in VLDL-sphingomyelin after 7 d could relate to the exchange of sphingomyelin between lipoproteins.
Plasma proteins capable of such transfer have been identified
(Tall et al. 1985
). When the concentration of
VLDL-sphingomyelin was high in the casein-fed rats, the LDL-
and HDL-2-sphingomyelin concentrations were low. This might be caused
by sphingomyelin transfer between lipoproteins. However, the
casein-induced fall in LDL-sphingomyelin, and also the lowering
of group mean LDL cholesterol, may be caused by a decrease in the
density of the LDL particles so that they are in part recovered in the
VLDL fraction. Such a phenomenon explains the decrease in LDL
cholesterol in cholesterol-fed rats (Beynen et al. 1984
). Possibly, plasma transfer of sphingomyelin from HDL-2 to
VLDL may contribute to the increase in VLDL-sphingomyelin in the
casein-fed rats.
In conclusion, feeding of the casein-containing diet probably
resulted in an increased output of sphingomyelin by the liver as
reflected by raised levels of VLDL sphingomyelin. Sphingomyelin may be
an essential structural component of VLDL (Merrill and Jones 1990
) so that an increase in VLDL secretion, as occurs in
casein-fed rats (Beynen 1990
), is associated with
increased sphingomyelin secretion. To meet the extra demand for
sphingomyelin, the rate of sphingomyelin formation was enhanced by
up-regulation of the activities of sphingomyelin-synthesizing
enzymes, and the rate of its catabolism was lowered by
down-regulation of the activity of lysosomal sphingomyelinase.
Transfer of sphingomyelin from HDL-2 to VLDL particles might have
contributed to the high level of VLDL-sphingomyelin in the
casein-fed rats. The effects of casein feeding were transient, but
the mechanism underlying the adaptive response to a change in
sphingomyelin metabolism is unknown.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Manuscript received April 22, 1999. Initial review completed June 12, 1999. Revision accepted July 29, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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