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Laboratory of Veterinary Biochemistry and * Department of Laboratory Animal Science, Graduate School of Animal Health, Faculty of Veterinary Medicine, Utrecht University, Utrecht, The Netherlands
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • sphingomyelin • dietary pectin • liver • lipoproteins • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). There is considerable evidence for a positive correlation
between the tissue and plasma contents of sphingomyelin and cholesterol
in normal (Patton 1970
) and pathological conditions, including
atherosclerosis (Barenholz and Gatt 1982
, Böttcher and van Gent 1961
) and diseases such as Niemann-Pick and cancer (Barenholz and Gatt 1982
). In addition, there appears to be a coordinate regulation of
cholesterol and sphingomyelin metabolism. Sphingomyelin has been
reported to alter cholesterol metabolism and, consequently, to affect
cholesterol balance in the cell. An elevation of the sphingomyelin
content of fibroblasts resulted in enhanced cholesterol synthesis (Gatt and Bierman 1980
, Kudchodkar et al. 1983
). On the other hand, increased
hydrolysis of plasma membrane sphingomyelin went together with the
down-regulation of cholesterol synthesis and increased cholesterol
esterification in human fibroblasts (Chatterjee 1994
, Gupta and Rudney 1991
, Slotte and Bierman 1988
). However, ceramides, the degradation
products of sphingomyelin, inhibited acyl-CoA:cholesterol
acyltransferase in Chinese hamster ovary cells (Ridgway 1995
).
Cholesterol in turn, influences sphingomyelin metabolism. Dietary
cholesterol supplementation (1 g/100 g) for 1–3 wk increased both very
low density lipoproteins
(VLDL)4
cholesterol and sphingomyelin concentrations in plasma of rats (Geelen et al. 1995
). After feeding a cholesterol-enriched (3%) diet to rats,
hepatic sphingomyelin synthesis was stimulated after 10 d by
activation of phosphatidylcholine:ceramide phosphocholine transferase
(PC:cer-Pch transferase) and phosphatidylethanolamine:ceramide
phosphoethanolamine transferase (PE:cer-Pet transferase)
(Nikolova-Karakashian et al. 1992
), whereas degradation of
sphingomyelin via neutral or acidic sphingomyelinase was inhibited
(Geelen et al. 1995
, Nikolova-Karakashian et al. 1992
).
Ingestion of the gel-forming fiber pectin lowers the concentration of
cholesterol, both in liver and in plasma (Fernandez 1995
). The effect
of dietary pectin on sphingomyelin metabolism is unknown, but because
there is a direct correlation between cholesterol and sphingomyelin
concentrations, it is hypothesized that pectin consumption lowers VLDL
sphingomyelin. Changes in VLDL sphingomyelin concentrations may be due
to changes in hepatic sphingomyelin synthesis and/or catabolism.
In the present study the effect of dietary pectin on sphingomyelin metabolism was investigated. Rats were fed diets containing either pectin or sucrose and the concentrations of sphingomyelin in liver and plasma lipoproteins were measured. To examine the effect of pectin on sphingomyelin synthesis and catabolism, the activities of hepatic serine palmitoyltransferase (SPT), PC:cer-Pch transferase, PE:cer-Pet transferase and acidic and neutral sphingomyelinase were determined.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Chemicals.
C6-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 4 wk, were used. They were housed in groups of three
per cage in an animal room with a 12 h light:dark cycle (lights
on, 0700–1900 h). All rats were fed a cholesterol-enriched control
diet for 7 d. The composition of the diet was as follows (g/100
g): casein, 21; corn oil, 5; coconut fat, 15; cholesterol, 1; corn
starch, 22.1; sucrose, 27; 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. The
composition of the mineral and vitamin premixes has been described by
Verbeek et al. (1993)
. After 7 d (Day 0 of the experiment) the
rats were divided into two groups of 18 rats each and one group of six
rats, which were stratified by body weight. One group of 18 rats
continued to receive the control diet and the other group of 18 rats
was transferred to the same diet supplemented with 20 g citrus
pectin/100 g diet at the expense of sucrose. Both control and pectin
diets were in powdered form. Animals had free access to food and tap
water.
Collection and preparation of samples.
Samples were taken on Day 0 from the group of 6 rats, and on Days 7, 14, and 21 from six rats of each dietary group between 0900 and 1100 h without prior food deprivation. The animals were anesthetized with diethylether, and blood samples were drawn from the abdominal aorta and collected in heparinized tubes. The liver was excised and kept in ice-cold physiological saline until homogenization, which was performed within 1 h.
Lipoproteins were isolated from fresh plasma by density gradient
centrifugation (Terpstra et al. 1981
). On the basis of their density
(d, kg/L), VLDL (d < 1.006), low density
lipoproteins (LDL) (1.019 < d < 1.063), and
HDL-2 (1.063 < d < 1.125) were collected.
Isolated lipoprotein fractions were frozen and stored at -20°C until
further analysis.
Several pieces of liver were homogenized separately and used for lipid extraction and subcellular fractionation. A piece of liver (~1 g) was minced in nine volumes of a homogenization buffer containing 0.25 mol sucrose/L and 10 mmol Tris-HCl/L (pH 7.5). The tissue was homogenized with three pulses of 15 s with an Ultra turrax homogenizer (Janke and Kunkel, Staufen, Germany). The whole liver homogenate samples were frozen in aliquots in liquid nitrogen and stored at -70°C until use.
To isolate hepatic plasma membranes, a piece of liver (~3 g) was
minced in 4 volumes of a buffer containing 0.25 mol sucrose/L, 1 mmol
MgCl2/L, and 5 mmolTris-HCl/L (pH 7.4) and homogenized with
10 strokes of a loose-fitting Dounce homogenizer (type A; Kontes,
Vineland, NJ). Plasma membranes were isolated as described by Hubbard et al. (1983)
. After the last centrifugation step, the isolated plasma
membranes were washed by centrifugation at 10,000 x g
for 10 min. The pellet was resuspended in 1 mL 10 mmol Tris-HCl/L (pH
7.4) and stored at -20°C.
Microsomes were isolated from liver pieces by ultracentrifugation
according to Williams et al. (1984)
. The washed microsomes obtained
from 1 g liver were resuspended in 1 mL of a buffer consisting of
50 mmol HEPES/L (pH 7.4), 5 mmol dithiothreitol/L (DTT), 5 mmol EDTA/L,
and 200 g glycerol/L. Microsomes were frozen in liquid nitrogen
and stored at -70°C.
Extraction and analysis of cholesterol and phospholipids.
Hepatic cholesterol was extracted and analyzed by the method of Abell et al. (1952)
. Total cholesterol in whole plasma and lipoproteins was
determined enzymatically with a test kit (MPR CHOD-PAP method)
purchased from Boehringer Mannheim, Germany.
Phospholipids were extracted from whole liver homogenate, hepatic
plasma membranes, or lipoproteins by the procedure of Bligh and Dyer (1959)
. Phospholipids were separated by thin layer chromatography (TLC)
on silica 60G plates in a solvent system of chloroform:methanol:acetic
acid:water 50:25:4:1 (v/v/v/v). Spots were visualized with iodine vapor
and identified by comparison to known standards. The silica spots
corresponding to sphingomyelin were scraped off the plate and
quantified by phosphorus analysis (Rouser et al. 1966
).
Enzyme assays.
Determination of SPT (EC 2.3.1.50) activity was based on the
incorporation of [3H]serine into chloroform-soluble
products. Radiolabeled serine was purified by dissolving
[3H]serine in water and extracting with
chloroform:methanol 1:2 (v/v) as described by Bligh and Dyer (1959)
.
Microsomes were diluted in 50 mmolHEPES/L (pH 7.4), 5 mmol EDTA/L, and
5 mmol DTT/L to a protein concentration of ~2.5 g/L. The SPT activity
in ~50 µg microsomal protein was measured as described by Williams et al. (1984)
, except that the chloroform phase was washed six times
with 10 g NaCl/L. Dihydrosphingosine was used as the carrier.
The assays for PC:cer-Pch transferase and PE:cer-Pet transferase
activity were modified from Vos et al. (1995)
. Briefly, the assay
mixture contained, in a total volume of 250 µL, 26 µmol
C6-NBD ceramide/L, 174 µmol egg phosphatidylcholine or
phosphatidylethanolamine/L, 50 mmol Tris-HCl/L (pH 7.4), 5 mmol EDTA/L,
20 µmol Triton X-100/L, and 50 µL purified hepatic plasma
membranes. Control experiments demonstrated that the assays were linear
with protein up to 
200 µg of plasma membrane protein and with time
for 3 h. For routine purposes, the enzyme reaction was carried out
for 60 min at 37°C with ~125 µg of plasma membrane protein. The
reaction was stopped by adding 2 mL chloroform:methanol 1:1 (v/v).
Phase separation was obtained by the addition of 550 µL of 10 mmol/L
acetic acid/8.8 g/L KCl, and lipids were extracted. After drying the
chloroform phases, lipids were resolved by TLC on silica 60G plates in
chloroform:methanol:250 g ammonium hydroxide/L:water 70:30:4:1
(v/v/v/v) as the developing solvent. NBD-sphingomyelin spots were
detected under UV-light and scraped from the plates. The
NBD-sphingomyelin was extracted from the silica with
chloroform:methanol:water 1:2.2:1. For quantification, the
NBD-sphingomyelin was excited at 465 nm and its fluorescence measured
at 530 nm. The fluorimetry was carried out with a Perkin Elmer
Luminescence Spectrometer LS-50.
Acid sphingomyelinase (EC 3.1.4.12) was determined by measuring the
release of phospho[methyl-14C]choline from
[choline-methyl-14C]sphingomyelin. The assay was
performed essentially as described by Geelen et al. (1995)
with minor
modifications. The 100 µL reaction mixture contained 0.5 mmol
[14C]sphingomyelin/L (17 Bq/nmol), 1.5 mmol
taurodeoxycholate/L, 50 mmol sodium acetate/L (pH 4.4), and whole liver
homogenate corresponding to 125 µg protein. The assay was conducted
for 60 min at 37°C. The reaction was terminated by adding 2 mL
chloroform:methanol 1:1 (v/v). The addition of 700 µL of 10 g
NaCl/L yielded a separation of aqueous and organic phases. The
chloroform phase was removed and the water:methanol phase was washed
three times with 1 mL chloroform. The upper phase was collected and
evaporated to dryness under nitrogen. Radioactivity was measured by
liquid scintillation counting.
Neutral sphingomyelinase activity was determined in isolated plasma membranes. The 100 µL incubation mixture contained 0.5 mmol [14C]sphingomyelin/L (67 Bq/nmol), 1.5 mmol taurodeoxycholate/L, 30 mmol Tris-HCl/L (pH 7.4), 25 mmolMgCl2/L, and 30 µL isolated plasma membranes (~75 µg protein). The reaction was carried out for 20 min at 37°C. Subsequently, the same procedure was used as described above for acidic sphingomyelinase.
Protein determination.
Protein was measured by the method of Lowry et al. (1951)
with bovine
serum albumin as a standard.
Statistical analysis.
Results are given as the mean ± 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 data for Days 7, 14, and 21 were also subjected to ANOVA to disclose any diet effects. If the data were not normally distributed, even after logarithmic transformation, or the variances were not homogeneous, the Kruskal-Wallis test was used instead of ANOVA. The level of significance was pre-set at P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Body weights of the control and pectin-fed rats did not differ at Day 21 of the experiment (185 ± 13 and 184 ± 12 g, respectively; mean ± SD, n = 6), but relative liver weights of the pectin-fed rats were lower (4.55 ± 0.15 and 5.18 ± 0.20 g/100 g body weight, respectively; P < 0.001). Because of the lower content of metabolic energy of the pectin-containing diet, the food intake of rats fed this diet was about twice that of rats fed the control diet.
Cholesterol concentrations in liver, whole plasma, and VLDL.
In rats fed the control diet, the concentration of hepatic cholesterol
rose significantly (P = 0.046) after Day 7. Consumption
of pectin significantly counteracted this rise in the concentration of
total cholesterol in liver (Fig. 1A
). Compared to controls, rats fed dietary pectin had a significantly
lower concentration of total cholesterol in whole plasma on Days 14 and
21 (Fig. 1
B). Fractionation of plasma lipoproteins revealed
that the pectin-induced lowering of total cholesterol in plasma was
caused by a reduction in VLDL cholesterol (Table 1). After 14 d of feeding, the VLDL cholesterol in pectin-fed rats
was reduced by 79% compared to the control rats. There were no
significant differences in LDL and HDL-2 cholesterol concentrations
between pectin-fed and control rats (Table 1)
.
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Over the course of the experiment, sphingomyelin concentrations in
either total liver homogenate or isolated plasma membranes were not
affected by feeding pectin. On Day 21 the concentrations were 6.0 ± 0.8 vs. 5.7 ± 0.5 and 30.7 ± 2.3 vs. 31.4 ± 2.9
nmol/mg protein for homogenate and plasma membranes from controls and
pectin-fed rats, respectively (mean ± SD, n= 6). Dietary pectin did not affect whole plasma sphingomyelin,
but altered sphingomyelin levels in plasma lipoproteins (Fig. 2). After 21 d of pectin consumption, sphingomyelin concentration in
VLDL was diminished by 57 % in pectin-fed rats compared to controls
(Fig. 2
B). The amount of sphingomyelin in the HDL-2 fraction
was enhanced by pectin on Days 14 (P < 0.001) and 21
(P = 0.1) of the experiment. The dietary effect was
significant when the data were analyzed by ANOVA. LDL sphingomyelin
levels were significantly greater in pectin-fed rats on Day 21.
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Over the course of the experiment, hepatic SPT activity of the control
and pectin-fed rats did not differ significantly; on Day 21 the
activities were 8.8 ± 4.2 and 8.1 ± 3.2 pmol/min ·
mg-1 protein for control and pectin-fedrats,
respectively (mean ± SD, n = 6). Moreover,
the activitiesof the other synthesizing enzymes, PC:cer-Pch
transferase and PE:cer-Pet transferase, was either not affected or not
affected systematically by the pectin diet (Fig. 3).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). In the present study, with rats fed a
cholesterol-enriched control or experimental diet, the pectin-induced
reduction of plasma cholesterol was caused by a decrease in VLDL
cholesterol. The new finding is that dietary pectin affects
sphingomyelin metabolism. Pectin did not influence the activity of the
enzymes involved in sphingomyelin synthesis, SPT and PC:cer-Pch
transferase, but it increased in sphingomyelin degradation as indicated
by activation of acidic and neutral sphingomyelinase. Furthermore,
pectin feeding lowered the level of sphingomyelin in VLDL, suggesting
that secretion of VLDL from the liver may be reduced.
After long-term pectin feeding, sphingomyelin metabolism should reach a
new steady-state in which hepatic and plasma sphingomyelin pools are
kept constant by equal input and output. An attempt to describe the new
steady state is schematically illustrated in Fig. 5
. After the consumption of pectin, both acidic and neutral
sphingomyelinases in the liver were activated, but sphingomyelin
synthesis was not altered. The enhanced degradation of sphingomyelin
was associated with an unchanged hepatic sphingomyelin level. Thus, the
liver must have compensated for the increase in sphingomyelin
degradation by the reduction of secretion and/or enhanced sphingomyelin
uptake. Although there was a drop of VLDL sphingomyelin levels, there
was probably no decrease in the hepatic secretion of sphingomyelin, as
studies with hepatocytes in culture have revealed that secreted VLDL
particles contain a substantial amount of ceramide but not much
sphingomyelin (Merrill et al. 1995
). It seems that the pectin-induced
low level of VLDL caused either a low uptake of sphingomyelin, while
this lipoprotein is being circulated, or a low conversion of the
ceramide in the lipoprotein into sphingomyelin. Whether pectin reduces
sphingomyelin secretion from the liver into bile is not known. The
uptake of sphingomyelin by the liver is probably enhanced after pectin
feeding. The pectin-induced decrease in the amount of liver cholesterol
will cause an upregulation of the expression of the LDL receptor (Brown and Goldstein 1981
), which may lead to enhanced uptake of LDL
sphingomyelin.
|
We can only speculate as to the mechanism by which dietary pectin
enhances the activities of the sphingomyelinases. Raising hepatic
cholesterol levels by dietary cholesterol challenge caused inhibition
of acidic (Geelen et al. 1995
) and neutral (Nikolova-Karakashian et al. 1992
) sphingomyelinase. Under in vitro conditions, sphingomyelinase
from human fibroblasts (Maziere et al. 1981
) or from rat liver (Geelen et al. 1995
) was inhibited by cholesterol. We found that lower
cholesterol concentrations in the liver of the pectin-fed rats
coincided with enhanced activities of acidic and neutral
sphingomyelinase (Fig. 4)
. Thus, it seems that the pectin-induced
lowering of hepatic cholesterol in turn relieved the inhibition of the
sphingomyelinases (Fig. 5)
.
Reduction of VLDL secretion by the liver under conditions of lowered
hepatic cholesterol levels is in agreement with the regulation of
hepatic secretion of VLDL by cholesterol (Fungwe et al. 1992
, Meijer et al. 1992
). Feeding cholesterol resulted in the accumulation of
cholesterol esters in the liver and stimulation of VLDL secretion,
which is most likely secondary to the increment of the hepatic
cholesterol pool (Fungwe et al. 1992
, Meijer et al. 1992
). Conversely,
when lovastatin, an inhibitor of cholesterol synthesis, was given to
rats, secretion of VLDL by the liver was diminished (Khan et al. 1989 and 1990
). Also in the present study, the reduction in hepatic
cholesterol concentration was accompanied by a drop in the cholesterol
concentration in the plasma VLDL fraction.
We assume that the pectin-mediated decrease in VLDL sphingomyelin concentration was secondary to the lowering of plasma VLDL as caused by a fall in VLDL secretion by the liver. The questions then arise how dietary pectin inhibits VLDL secretion, and why a low level of VLDL results in a low VLDL sphingomyelin concentration. Dietary pectin counteracted the rise in liver cholesterol that was caused by the high cholesterol intake by the rats. As cholesterol regulates VLDL secretion, the pectin-induced lowering of hepatic cholesterol concentration may have directly reduced VLDL secretion. A decrease in VLDL secretion as mediated by pectin feeding will be associated with a decrease in the uptake of sphingomyelin by circulating VLDL particles or be associated with a decrease in sphingomyelin synthesis within these lipoproteins, thus resulting in lowered VLDL sphingomyelin concentrations in plasma.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported in part by the Netherlands Foundation
for Chemical Research (SON) with financial aid from the Netherlands
Organization for Scientific Research (NWO).
2 The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
4 Abbreviations used: DTT, dithiothreitol; HDL,
high density lipoproteins; LDL, low density lipoproteins; PC:cer-Pch
transferase, phosphatidylcholine:ceramide phosphocholine transferase;
PE:cer-Pet transferase, phosphatidylethanolamine:ceramide
phosphoethanolamine transferase; SPT, serine palmitoyltransferase; TLC,
thin layer chromatography; VLDL, very low density lipoproteins.
Manuscript received August 11, 1998. Initial review completed October 2, 1998. Revision accepted November 24, 1998.
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