(Received for publication, October 31, 1994; and in revised form, March 27, 1995 )
From the
Sphingolipids are constituents of liver and lipoproteins, but
relatively little is known about their synthesis and secretion.
Incubation of rat hepatocytes with [C]- or
[
H]serine labeled the long-chain base backbones
of mainly ceramide and sphingomyelin. Most of the labeled sphingolipids
were associated with the cells; however, 1-5% (the majority of
which was ceramide) was released into the medium as part of very low
density lipoproteins (VLDL). Since this is the first report that
lipoproteins contain ceramide, lipoproteins were isolated from rat
plasma, and the ceramide contents were (per mg of protein): 6.5 nmol
for VLDL (d < 1.018), 0.6 nmol for low density lipoproteins
(1.018 < d < 1.063), 0.2 nmol for high density
lipoproteins (1.063 < d < 1.18), and 0.1 nmol for the
albumin fraction; the lipoproteins also contained 0.1-0.4 nmol of
free sphingosine/mg of protein. A number of factors affected the
secretion of radiolabeled sphingolipids: 1) addition of palmitic acid,
but not stearic or oleic acid, enhanced secretion due to an increase in
long-chain base synthesis de novo. 2) Choline deficiency
caused a 42% reduction in the secretion of radiolabeled sphingomyelin,
but this was due to an effect on VLDL secretion rather than a decrease
in sphingolipid synthesis. Removal of choline was examined because
previous studies (Yao, Z. M., and Vance, D. E.(1988) J. Biol. Chem. 263, 2998-3004) have shown that choline deficiencies depress
[Abstract]
phosphatidylcholine synthesis and lipoprotein secretion. 3) Incubation
of the cells with fumonisin B
, a mycotoxin inhibitor of
sphinganine (sphingosine) N-acyltransferase, reduced overall
sphingolipid synthesis and secretion by 90%, but had no effect on the
secretion of apoB, phosphatidylcholine, or cholesterol. All together,
these findings demonstrate that rat hepatocytes synthesize ceramide and
sphingomyelin de novo and incorporate them into both cellular
membranes and secreted VLDL, but normal sphingolipid synthesis is not
required for lipoprotein secretion.
Sphingolipids are constituents of cellular membranes, lipoproteins, secretions such as lung surfactant and milk, and the water barrier of skin (Sweeley, 1991). They are composed of a long-chain (sphingoid) base with an amide-linked fatty acid and, except in the case of free ceramide, polar headgroups such as phosphorylcholine (for sphingomyelin) or carbohydrates (for cerebrosides, gangliosides, and other complex glycolipids). Sphingolipids have a wide range of functions, from structural roles to modulation of growth factor receptors and signal transduction pathways (for reviews see Bell et al.(1993)). There have been relatively few studies of the de novo biosynthesis of the lipid backbones of sphingolipids in intact cells and how this may relate to the emerging picture of these compounds as bioactive species.
Sphingolipid biosynthesis begins with the synthesis of 3-ketosphinganine by condensation of serine and a fatty acyl-CoA, followed by reduction of the keto group using NADPH (producing sphinganine) and addition of an amide-linked fatty acid (Merrill and Wang, 1986). The resulting N-acylsphinganines (dihydroceramides) are further metabolized to ceramides (i.e. containing a 4,5-trans-double bond in the sphingosine group of the ceramide) (Merrill and Wang, 1986; Rother et al., 1992) with the various headgroups (for reviews of sphingolipid metabolism, see Merrill and Jones(1990); Sweeley, 1991; Kolesnick, 1991; van Echten and Sandhoff, 1993).
The de novo pathway
can be followed by incubation of cells with
[C]serine (or labeled palmitic acid) and
incorporation of label in the sphinganine and sphingosine backbones of
various sphingolipids (Merrill and Wang, 1986). Flux through this
pathway is affected by the concentration of serine and palmitate in the
culture media (Merrill et al., 1988a; Messmer et al.,
1989); therefore, precursor availability appears to be one of the
determinants of the rate of sphingolipid biosynthesis de novo (Merrill and Jones, 1990). Regulation also appears to involve
inhibition by free sphingosine, which decreases the activity of serine
palmitoyltransferase (Van Echten et al., 1990).
Sphingomyelin and some glycolipids are major constituents of plasma
(Vance et al., 1975) and circulating lipoproteins (see review
by Merrill and Jones(1990)). Some gangliosides are known to be shed by
tumors and to become associated with serum lipoproteins (Valentino and
Ladisch, 1992) and gangliosides appear in liver perfusates (Kivatinitz
et al., 1992); nonetheless it is not yet known if
sphingolipids that are synthesized by liver are secreted or merely bind
to lipoproteins while they are in circulation. Sphingolipid synthesis
from [C]serine has been investigated using rat
hepatocytes and a liver cell line (Messmer et al., 1989);
however, this study did not characterize the nature of the complex
sphingolipids that were made, nor whether they were secreted from the
hepatocytes with lipoproteins. Hence, the goals of this investigation
were to provide this basic information about the composition and fate
of the sphingolipids made de novo by rat hepatocytes and to
explore the implications of these findings for the regulation of
sphingolipid (and lipoprotein) biosynthesis and secretion.
To compare the
composition of the ceramides that were produced by the cells versus those released into the medium, cells were labeled overnight with
0.1 mCi of [H]serine/dish, then the media from 10
dishes were pooled, and the cells were scraped from each dish. The
medium was centrifuged to remove dislodged cells and analyzed without
further purification, because essentially all of the radiolabeled
sphingolipids in the medium were found to be associated with
lipoproteins (as will be shown under ``Results''). The lipids
in the cells and medium were extracted, base-hydrolyzed in 0.5 N KOH in
methanol for 1 h at 37 °C, and the ceramides purified by
preparative TLC using silica gel plates and the solvent system diethyl
ether:methanol (99:1, v/v). To recover the ceramides, silica from the
appropriate region of the TLC plates was scraped into test tubes and
extracted with 1 ml of chloroform:methanol (1:1, v/v) followed by 1 ml
of methanol. The [
H]ceramide samples were
analyzed by HPLC using a Waters C
-µBondapak column and
acetonitrile:ethanol (60:40, v/v) as the mobile phase. The radiolabel
was detected using a
-Ram flow-through monitor.
To produce
[H]ceramides as standards for the HPLC analyses,
various fatty acyl-CoAs were incubated with
[
H]sphingosine as described by Merrill and
Wang(1992), and the products were analyzed as described above (the
retention times of the various
N-acyl-[
H]sphingosines are given in
Fig. 5
).
Figure 5:
Analyses of
[H]ceramides in cells (middle panel) and
medium (lower panel) after incubation of hepatocytes with
[
H]serine. Hepatocytes (3-4 mg/dish) were
incubated overnight with 100 µCi of
[
H]serine, then the
[
H]ceramides were extracted and purified by TLC
from the cells (two dishes were pooled) and the culture medium (10
dishes were pooled) as described in the text. The
[
H]ceramides were separated by reverse-phase HPLC
(Waters C
-µBondapak column) with acetonitrile:methanol
(60:40, v/v) as the eluting solvent) and quantitated with an in-line
radioactivity detector. Standard [
H]ceramides
were prepared by incubating [
H]sphingosine with
different fatty acyl-CoA's and rat liver microsomes (as described
in Merrill & Wang, 1992); the retention times for ceramides with
fatty acids of varying chain length are shown in the upper
panel.
To determine the effect of
fumonisin B on the secretion of apoB, hepatocytes were
isolated as described above and on the following morning changed to new
medium minus serum and with or without 10 µM fumonisin
B
. After 8 and 24 h, the medium was removed, centrifuged to
remove debris, and the apoB was analyzed by enzyme-linked immunosorbent
assay as described in Yao and Vance(1988, 1990).
An earlier study (Messmer et al., 1989) showed that
hepatocytes incorporate [C]serine into
sphingolipids without accumulation of detectable amounts of the free
long-chain base intermediates. The goals of this investigation were to
characterize the complex sphingolipids that are made using these newly
synthesized long-chain bases, to determine if hepatocytes release newly
made sphingolipids as part of secreted lipoproteins, and to identify
some of the factors that regulate the de novo biosynthesis and
secretion of sphingolipids.
The time course of sphingolipid
formation from [C]serine is given in
Fig. 1
. For the first 6 h, essentially all of the radiolabel
could be accounted for by ceramide and sphingomyelin. Sphingosine was
the major long-chain base of the total and individual sphingolipids,
and this did not vary with time or any of the treatments in this study
(except fumonisin addition); therefore, the results are presented as
the total long-chain bases, which is the sum of radiolabel in
sphingosine and sphinganine.
Figure 1:
Time course of sphingolipid labeling
from [C]serine. Hepatocytes (approximately 2.7
mg of protein/culture dish) were incubated with 20 µCi of
[
C]serine for the times shown, then the lipids
were extracted as described in the text. Aliquots of the lipid extract
were acid-hydrolyzed to sphingosine and sphinganine (for quantitation
of the total sphingolipids) or separated on TLC (for ceramide and
sphingomyelin) followed by quantitation of the radiolabel in the
relevant regions of the TLC plates. After 4 h, 10 mM serine
was added to some of the dishes (designated Chase in
B), and the sphingolipids were analyzed at the times shown.
The data are given as the mean ± S.E. (n = 4) in
total sphingolipids (closed circles), ceramide (open
squares), and sphingomyelin (closed
squares).
After 4 h, ceramide appears to reach a
steady state; whereas, the labeling of sphingomyelin and other complex
sphingolipids continued for at least 12 h (Fig. 1A).
Addition of an unlabeled serine ``chase'' at 4 h
(Fig. 1B) stopped the formation of labeled sphingomyelin
and total sphingolipids. That [C]ceramide
remained relatively constant during both continuous labeling and the
chase implies that ceramide is not only an intermediate in the
formation of more complex sphingolipids, but also, itself a relatively
significant product of the de novo biosynthetic pathway. Based
on the specific activity of the added [
C]serine,
the cellular level of [
C]ceramide is at least
0.3 nmol/mg of protein after 4 h of labeling. A more exact estimate of
the amount of [
C]ceramide is precluded by the
dilution of the [
C]serine by serine made from
various unlabeled precursors, such as glycine (via serine
hydroxymethyltransferase) and glucose (via intermediates of
glycolysis). In fact, the equilibration of serine and glycine was so
rapid that almost as many disintegrations/min were incorporated into
sphingolipids when hepatocytes were incubated with
[
C]glycine of a comparable specific activity
(data not shown).
By 12 h, about one-third of the radiolabel was
distributed among many minor bands that were too faint to be readily
distinguished. When 10-fold higher amounts of
[C]serine (100 µCi/dish) were used (not
shown), one of these could be identified as lactosylceramide, but most
of these products were still too faint to be analyzed readily. It is
known that the glycolipid pattern changes while rat hepatocytes are in
culture (Phillips et al., 1985); therefore, this study focused
on the incorporation of [
C]serine into ceramide,
sphingomyelin, and total sphingolipids.
Figure 2:
Time course of sphingolipid labeling from
[C]serine and recovery in the cells (A)
or culture medium (B). Hepatocytes (approximately 4.3 mg of
protein/culture dish) were incubated with 20 µCi of
[
C]serine and 0.4 mM palmitate:BSA
(prepared as the 2:1 molar ratio, i.e. 0.8 mM in
palmitate) for the times shown, then the medium was removed from the
cells, and the cells were washed gently with PBS. The medium and PBS
washes were centrifuged in a bench top centrifuge to remove any
dislodged cells, then the medium and cells were separately extracted
and analyzed for total sphingolipids (Total SL, closed
circles), ceramide (Cer, open squared), and sphingomyelin
(SM, closed squares) as described in the legend to Fig. 1 and
the text. The medium from four dishes was pooled to increase the number
of disintegrations/min; however, the data are given in the
disintegrations/min/dish. The data are given as the mean ± S.D.
for triplicate analyses.
To
determine the nature of the particles that contain these radiolabeled
sphingolipids, cells were incubated with
[H]serine to increase the amount of label in the
medium. After 12 h, the medium was removed, centrifuged to remove
cells, and subjected to sucrose gradient centrifugation (Fig. 3).
The majority of the radiolabel in both total sphingolipids and
sphingomyelin was recovered in the fractions with d <
1.055, which contains VLDL. A small amount (primarily sphingomyelin)
was in the most dense fractions, which contain albumin.
Figure 3:
Amounts of labeled sphingolipids in
culture media after separation by density. Hepatocytes (approximately
2.7 mg of protein/culture dish) were incubated with 200 µCi of
[H]serine and 0.4 mM palmitate:BSA
(prepared as the 2:1 molar ratio) for 12 h, then the medium was removed
and centrifuged for several minutes at approximately 8,000
g to remove any dislodged cells. The medium was then subjected
to sucrose-density gradient centrifugation for 18 h as described in the
text, different fractions were collected, and the lipids were extracted
and analyzed for total sphingolipids (Total SL, closed
circles) and sphingomyelin (SM, closed squares) as
described in the legend to Fig. 1 and the text. The densities of the
fractions were determined by weighing a precise volume of each fraction
and are presented in the upper panel as g/ml. Shown are
representative data from triplicate analyses that gave similar results,
but have not been combined because of small differences in the
gradients from different tubes.
A similar
analysis was conducted with both [C]serine
(Fig. 4, left panel) and [
H]serine
(Fig. 4, right panel) and separation of the lipoproteins
by sequential centrifugation of the medium at different densities
achieved by addition of KBr. The majority of the radiolabel (>75%)
was recovered with the VLDL fractions. These findings establish that
most of the newly made sphingolipids that are released from the
hepatocytes are associated with VLDL rather than membrane fragments or
HDL. The selective association with VLDL makes it likely that these
sphingolipids are secreted as part of nascent VLDL.
Figure 4:
Quantitation of the labeled sphingolipids
in different lipoprotein fractions. Hepatocytes (approximately 3.4
mg/dish) were incubated with 200 µCi of
[C]serine (left panel) or 0.5 mCi of
[
H]serine (right panel) and 0.4
mM palmitate:BSA (prepared as the 2:1 molar ratio) for 12 h,
then the medium was removed and centrifuged for several minutes at
approximately 8,000
g to remove any dislodged cells.
The medium was then subjected to sequential centrifugation at densities
for VLDL (d < 1.018), LDL (1.018 < d <
1.063), and HDL (1.063 < d < 1.18). The lipids were
extracted, acid-hydrolyzed, and analyzed for total sphingolipids
(i.e. radiolabel in sphingosine and sphinganine). The left
panel represents the results from duplicate analyses
(±range) for [
C]serine and a single
sample incubated with [
H]serine. The
disintegrations/min are given for the lipids from the entire
dish.
To determine if the ceramides secreted by the hepatocytes are similar in structure to the ones found in the cells, the ceramide fraction was analyzed by reverse-phase HPLC (Fig. 5). The HPLC profiles for the cells (middle panel) and medium (lower panel) are generally similar, with most of the radiolabel eluting in the vicinity of ceramides with arachidic (C20), behenic (C22), and lignoceric (C24) acids (the multiple peaks eluting after C20 may be due to unsaturation of the fatty acid or differences in the long-chain base). This composition appears reasonable, because these chain lengths are present in >60% of the sphingomyelins from liver (Fex, 1971). There are two possible differences between the cells and medium: 1) N-palmitoyl-(C16) and N-stearoyl-(C18) sphingosines were evident in the cells but were not seen in the medium, although the amounts of total radiolabel in the medium may have been too low for these minor species to be detected, and 2) the ceramide(s) in the vicinity of N-lignoceroyl-sphingosine (C24) eluted slightly earlier for the medium than for the cells.
Preliminary analyses were also conducted with lymph that had been collected from rats that had an intestinal lymph fistula and given to us by Dr. Patrick Tso (Louisiana State University Medical Center, Shreveport, LA). The lymph contained approximately 1 nmol of total sphingolipids/ml, of which about 40% was ceramide. Therefore, these analyses establish that ceramide is one of the neutral lipid components of very low density lipoproteins as well as other lipoproteins to a lesser extent.
Figure 6:
Stimulation of sphingolipid biosynthesis
from [C]serine by palmitic acid. Hepatocytes
(approximately 2.5 mg/dish) were incubated with 20 µCi of
[
C]serine in DMEM alone or supplemented with 0.4
mM fatty acid-depleted bovine serum albumin (BSA) or the BSA
complex with oleic acid (18:1, BSA), palmitic acid (16:0, BSA), or
stearic acid (18:0, BSA) in the mol ratios shown (e.g. 2:1
palmitate:BSA represents 0.4 mM BSA with 0.8 mM
palmitic acid). After 4 h the lipids were extracted and analyzed for
total sphingolipids (Total SL, hatched bars) and sphingomyelin (SM,
solid bars) as described in the legend to Fig. 1 and the text. The data
are given as the mean ± S.D. for n =
3.
Another consequence of adding fatty acids to the cells was a
reduction (by about half) in the incorporation of label from
[C]serine into fatty acids (similar results were
obtained with palmitic, stearic, and oleic acids) (data not shown).
Because palmitic acid increases overall sphingolipid labeling, its
effect on the secretion of total sphingolipids and ceramide was
determined for the pooled lipoproteins secreted by hepatocytes
incubated with [C]serine (Fig. 6,
right panel). When the hepatocytes were incubated with
palmitate:BSA, the radiolabel in secreted sphingolipids was almost
2-fold higher than when BSA was added alone or when oleate:BSA was
added. Ceramide labeling was also approximately 2-fold higher when
palmitic acid was added than for oleic acid or BSA alone. Therefore,
the availability of this precursor of de novo sphingolipid
biosynthesis also affects the amount of secreted sphingolipid.
Somewhat surprisingly, there was an
approximately 2-fold increase in the appearance of radiolabel in
sphingomyelin when choline was restricted. It is possible that this is
arising from a second pathway that has been proposed for sphingomyelin
synthesis (Fig. 7) in which ceramide is first converted to
ceramide phosphorylethanolamine (via transfer of the headgroup from
phosphatidylethanolamine) (Muehlenberg et al., 1972; Malgat
et al., 1986) followed by methylation (Ullman and Radin,
1974). Rat liver has been reported to contain ceramide
phosphorylethanolamine (Muehlenberg et al., 1972), and we have
observed that [C]serine is incorporated into a
product with the same mobility on thin-layer chromatography as ceramide
phosphorylethanolamine (Fig. 7). The amounts were too small for
definitive structural identification; however, this compound should be
borne in mind for future study.
Figure 7:
Radiometric scan of the labeled
phosphosphingolipids from hepatocytes incubated with
[C]serine. Hepatocytes (approximately 2.7 mg of
protein/dish) were incubated with 20 µCi of
[
C]serine for 12 h, and the lipids cells were
extracted as described in the experiment in Fig. 1. After base cleavage
to remove the glycerolipids, the sphingolipids were separated by silica
gel chromatography with development of the TLC plates with
CHCl
:methanol:acetic acid:H
O (25:15:4:2, v/v).
After the plates were air-dried, the radiolabel was detected with a
BioScan radiometric detector. The identity of the SM was assigned by
comparison with a standard; the ceramide phosphorylethanolamine
(CPE) was identified as a ninhydrin-positive, base-stable
compound with the appropriate R based on the properties
reported by others (Muehlenberg et al., 1972; Malgat et
al., 1986). Shown above the scan are the pathways that have been
proposed for the synthesis of ceramide phosphorylethanolamine and
sphingomyelin by transfer of the phosphoheadgroup from
phosphatidylethanolamine (PE) and phosphatidylcholine
(PC), respectively, to ceramide (Cer) with the
liberation of diacylglycerol (DAG). Ceramide
phosphorylethanolamine can be methylated to SM using
S-adenosylmethionine (SAM) and producing
S-adenosylhomocysteine
(SAH).
In contrast to the lack of a reduction on total sphingolipid synthesis, the secretion of radiolabeled sphingolipids was reduced by about half in choline deficiency (Table II), which is similar to the reduction in VLDL secretion in this model (Yao and Vance, 1988; Vance, 1990). Therefore, although hepatocytes are capable of synthesis of sphingolipids in choline deficiency, their secretion into the medium is impaired, because fewer lipoproteins are secreted.
Analyses of
the radiolabel in the pooled lipoproteins (d < 1.18 g/mg)
(Table III) revealed that there was no reduction in the secretion
of labeled phosphatidylcholine or cholesterol, but total sphingolipids
were reduced by 92%, sphingomyelin by 37%, and ceramide by 95%.
Fumonisin B did not inhibit the secretion of apoB: after 8
h, the nanograms of apoB secreted per milligrams of cell protein was
222 ± 56 versus 172 ± 34 in the absence and
presence of 10 µM fumonisin B
, respectively;
after 24 h, the amounts were 324 ± 48 versus 304
± 34 in the absence and presence of 10 µM fumonisin
B
, respectively (n = 3). These results
establish that sphingolipid biosynthesis can be inhibited substantially
without having an effect on lipoprotein secretion by hepatocytes.
This report presents the first description of long-chain base
synthesis and secretion by isolated liver cells. As far as we are
aware, the only previous report of secretion of sphingolipids by
hepatocytes was the notation by Siutta-Mangano et al.(1982) of
labeled sphingomyelin in lipoproteins secreted by chicken hepatocytes
incubated with radiolabeled acetate, and this could have occurred by
remodeling of existing sphingolipids rather than de novo synthesis. In agreement with our previous study with hepatocytes
in short term culture (Messmer et al., 1989), radiolabeled
serine was incorporated into the sphingosine backbone of complex
sphingolipids. Somewhat surprisingly, a substantial portion of the
radiolabel remained in ceramide even after 12 h with or without a chase
with unlabeled serine. This suggests that ceramide per se is a
significant product of de novo sphingolipid biosynthesis. This
finding is particularly interesting considering the biological
activities that have been suggested for ceramide as a second messenger
(Kim et al., 1991; Dobrowsky and Hannun, 1992; Dressler et
al., 1992) in the action of tumor necrosis factor and other
cytokines. We have recently found that rat hepatocytes also respond to
short-chain ceramides (with induction of 1-acid glycoprotein and
suppression of CYP2C11) and that sphingomyelin hydrolysis is stimulated
by interleukin-1
,
(
)
which suggests that
ceramide may be utilized as a second messenger in hepatocytes as in a
number of cell types.
A portion of the radiolabeled sphingolipids
was secreted with VLDL, which establishes that at least some of the
sphingolipids that are found in lipoproteins are incorporated during
the biosynthesis and release of VLDL by hepatocytes. However, the
amount of sphingomyelin label and mass in VLDL was small; therefore,
most of the sphingomyelin that is in LDL must be coming from sources
other than synthesis within the hepatocyte. Since sphingomyelin is a
major constituent of red blood cells and the plasma membranes of most
tissues, it is likely that these are sources of the sphingomyelin of
LDL. Because the hepatocytes in culture incorporated little radiolabel
from [H]- or [
C]serine
into glycolipids, it is not possible to conclude whether liver is a
major source of glycosphingolipids in serum. From studies of perfused
liver, it appears that some serum gangliosides might arise from liver;
other studies of the incorporation of [
H]glucose
into plasma glycosphingolipids by humans (Vance et al., 1975)
have suggested that serum glycolipids can come from both liver and
other sources.
The finding of substantial amounts of labeled
ceramide in the secreted VLDL was corroborated by analyses of the
ceramide mass in lipoproteins isolated from rat plasma. It is
noteworthy that ceramide is found in substantial amounts in VLDL and
that there is 10-fold less in LDL. This suggests that the ceramide is
either removed or converted to another sphingolipid, such as
sphingomyelin, while in circulation. We have previously found that VLDL
decreased [C]serine incorporation into
long-chain bases by hepatocytes (Messmer et al., 1989). To a
lesser extent, LDL was also inhibitory, as has been seen previously
with other cell types (Verdery and Theolis, 1982; Merrill, 1983;
Chatterjee et al., 1986). The mechanism of this inhibition is
unknown; however, the finding that ceramide is a substantial component
of VLDL could mean that when this compound is taken up by cells, it is
utilized in place of endogenous synthesis for the formation of cellular
sphingolipids. It is also possible that lipoprotein sphingolipids are
hydrolyzed to sphingosine, which has been shown to suppress de novo sphingosine biosynthesis by decreasing the activity of serine
palmitoyltransferase (Van Echten et al., 1990).
It is likely that many of the factors that regulate de novo sphingolipid biosynthesis are still not known. Nonetheless, one factor that is important is the availability of the precursor palmitic acid, because stimulation by this fatty has now been seen in mouse L cells (Merrill et al., 1988a), hepatocytes (Messmer et al., 1989 and this study), and CaCo-2 cells (Chen et al., 1993). The selective stimulation of long-chain base formation by palmitic acid indicates that this pathway may be influenced by some of the many determinants of palmitic acid synthesis and turnover, including the amount and type of dietary fat. These interactions are complex because there is no stimulation, and perhaps some inhibition, by other fatty acids that are biosynthesized by liver (such as the stearic acid and oleic acid used in this study).
There is
considerable interest in the possibility that there is a relationship
between cholesterol and sphingomyelin metabolism (Barenholz and
Thompson, 1980; Barenholz and Gatt, 1982; Slotte et al.,
1990). For examples, SM correlates closely with the amounts of
cholesterol in different membranes (Patton, 1970), and there are
structural reasons for these two lipids to interact somewhat
preferentially (Vandenheuvel, 1965). The presence of SM influences
cholesterol movement among membranes (Lange et al., 1979;
Wattenberg and Silbert, 1983; Fugler et al., 1985; Bittman,
1988; Stein et al., 1988), including the distribution of
cholesterol among membranes (Yeagle and Young, 1986), and SM has also
been seen to alter the interaction of cholesterol with proteins
(Stevens et al., 1986). These lipids also appear to be linked
metabolically, as reflected in altered SM metabolism in
hypercholesterolemia (Maziere et al., 1984), the recent
findings that sphingomyelin (like cholesterol) is transported to the
plasma membrane via a pathway distinct from protein secretion (Shiao
and Vance, 1993) and that the turnover of sphingomyelin in response to
tumor necrosis factor- induces the synthesis of cholesteryl esters
(Chatterjee, 1994). Addition of SM to cells in culture affects LDL
binding and utilization, as well as altering cholesterol metabolism
(Gatt and Bierman, 1980; Kudchodkar et al., 1983). Thus, the
mechanisms for this association may be both physical (even if SM simply
presents a hydrophobic environment with a greater partition coefficient
for cholesterol) and metabolic. It is interesting, therefore, that the
incubation of the hepatocytes with fumonisin B
inhibited
de novo sphingomyelin synthesis but did not alter the
incorporation of radiolabel into cholesterol.
The finding that choline deficiency does not decrease sphingomyelin biosynthesis may indicate that sphingomyelin synthase requires only low amounts of phosphatidylcholine, which is the major precursor of the headgroup of sphingomyelin, either because it has a high affinity for this co-substrate, or because it is located in a subcellular compartment that is less affected in choline deficiency. Previous studies of sphingomyelin biosynthesis based on the amount of radiolabeled choline in the headgroup have also concluded that sphingomyelin continues to be made, even when choline is limiting (Hatch and Vance, 1992). In contrast, limiting ceramide appears to have a large effect on sphingomyelin synthesis (Merrill et al., 1993).
It has been
shown that phosphatidylcholine is required for secretion of VLDL;
however, it is not known whether or not sphingomyelin is also involved
in this process. Recent studies (Field et al., 1993) have
found that treatment of CaCo-2 cells with sphingomyelinase or
cell-permeable ceramides inhibited apoB synthesis and secretion;
however, these treatments may alter numerous properties of cells in
culture. When fumonisin B was used to inhibit de novo sphingolipid biosynthesis in this study, this mycotoxin had no
effect on either apoB secretion nor on the secretion of cholesterol or
phosphatidylcholine. Therefore, it is evident that in rat hepatocytes,
normal sphingolipid biosynthesis is not required for VLDL secretion.
>Table I: Sphingolipid content of rat lipoproteins
Lipoproteins were isolated from 11.7 ml of plasma. Total mg of protein for each lipoprotein class was: VLDL, 2.5 mg; LDL, 2.5 mg; HDL, 8.5 mg; and the more dense (albumin) fraction, 55 mg.
Table II: Sphingolipid biosynthesis by hepatocytes from choline deficient rats
For these experiments, hepatocytes were
isolated from choline deficient rats, then incubated overnight with
(+) or without (-) 28 µM choline and 100
µM methionine. The cells were then incubated with
[C]serine for 4 h, and the sphingolipids were
extracted and analyzed as described in the text. The values shown are
the mean ± S.D. for triplicate analyses.
The total sphingolipids were analyzed by acid
hydrolysis of the lipid extracts and quantitation of the radiolabel in
long-chain bases (without correction for recovery of the radiolabel,
which is generally around 50%). For ease of comparison, the approximate
amount of total sphingolipids using this correction factor has been
given in parentheses.(119)