(Received for publication, December 1, 1994; and in revised form, May 30, 1995)
From the
Long-chain (sphingoid) bases are highly bioactive intermediates
of sphingolipid metabolism, yet relatively little is known about how
the amounts of these compounds are regulated. This study used J774A.1
cells to characterize the ``burst'' of sphinganine and
sphingosine, or the transient increase of up to 10-fold in long-chain
base mass, that occurs when cells in culture are changed to fresh
medium. The increase in sphinganine was attributable to de novo sphingolipid biosynthesis because: 1) there is increased
incorporation of [H]serine and
[
H]palmitate into sphinganine; 2) the
incorporation of [
H]serine was equivalent to the
increase in sphinganine mass; 3)
-F-alanine, an inhibitor of
serine palmitoyltransferase, blocked the sphinganine burst; 4) the
magnitude of the burst depended on the concentration of serine in the
medium, which is known to affect long-chain base biosynthesis; and 5)
the appearance of sphinganine was relatively unaffected by
lyso-osmotrophic agents (NH
Cl and chloroquine) that blocked
sphingolipid hydrolysis in these cells. In contrast, the sphingosine
burst arose mainly from turnover of complex sphingolipids because no
incorporation of [
H]serine or
[
H]palmitate into sphingosine was detected;
sphingosine mass was not affected by
-F-alanine or the serine
concentration; and, the burst could be followed by the release of
sphingosine and ceramide from complex sphingolipids (especially
sphingomyelin) in a process that was inhibited by NH
Cl and
chloroquine. Additionally, the fate of these long-chain bases differed:
sphinganine was mostly (80-85%) acylated and incorporated into
dihydroceramide and complex sphingolipids, whereas most of the
sphingosine (70%) was phosphorylated and degraded, with incorporation
of the resulting ethanolamine phosphate into phosphatidylethanolamine.
Sphinganine, however, could be diverted toward degradation by adding an
inhibitor of N-acylation (fumonisin B
). In
accounting for the elevation in sphingosine and sphinganine after cells
are changed to new medium, these studies have provided fundamental
information about long-chain base metabolism. The existence of
differential changes in sphinganine and sphingosine, as well as their
1-phosphates and N-acyl-derivatives, should be considered when
evaluating the roles of sphingolipid metabolites in cell regulation.
Long-chain (sphingoid) bases have long been known to serve as
the backbones of sphingolipids(1) ; however, only recently have
they gained attention as bioactive compounds. Since the discovery that
sphingosine acts as a potent inhibitor of protein kinase C(2) ,
many other systems have been found to be affected by long-chain bases,
including the
Na,K
-ATPase(3) , phosphatidic
acid phosphatase(4, 5, 6, 7) ,
phospholipases (including phospholipase D)(8, 9) ,
retinoblastoma protein phosphorylation(10) , and
sphingosine-activated protein kinase(s)(11) . Furthermore, the N-acyl- (i.e. ceramide) (12, 13, 14) and
1-phosphate(15, 16, 17) derivatives of
sphingosine are highly bioactive and are emerging as mediators of
various cell signaling pathways (for recent reviews, see (12, 13, 14) , 18, 19).
Relatively little is known about the metabolism of long-chain bases as intermediates of de novo biosynthesis and the turnover of more complex sphingolipids. The amounts of free long-chain bases in cells are generally low (20, 21, 22) but can be affected by various factors (13, 20, 23) and toxins(24) . For example, treatment of growth arrested fibroblasts with platelet-derived growth factor increases sphingosine and sphingosine 1-phosphate, with the latter appearing to act as a mediator in the release of intracellular calcium (25) and activation of AP1(26) .
We have discovered that a common laboratory procedure, the changing of cells in culture to fresh medium, induces a transient ``burst'' of sphinganine and sphingosine in J774A.1 cells (27, 73, 74, 75) and Swiss 3T3 cells(28) . A similar observation has been made with several other cell types(29) , which suggests that this may be a common occurrence. The cellular levels of sphingosine that are achieved by adding fresh culture medium (approximately 0.5 nmol/mg protein) are similar to the amounts that affect cell function when sphingosine is added exogenously (18, 28, 30) . Therefore, this burst warrants further characterization to determine its potential relevance to the regulation of cell behavior by sphingolipids.
In this study, we establish that the free sphinganine of the burst arises from increased de novo sphingolipid biosynthesis, whereas the sphingosine is derived from sphingolipid turnover. The fate of the long-chain bases differed: although both can be converted to the 1-phosphates, sphinganine is mainly acylated, whereas sphingosine is degraded by J774A.1 cells. As far as we are aware, this study is the first to identify conclusively the origins and fates of these endogenous compounds in intact cells.
The D-erythro-sphingosine, DL-threo-sphingosine, ceramides, sphingomyelin, and
gangliosides were from Sigma; DL-erythro-sphinganine
was from United States Biochemical Corp. (Cleveland, OH);
3-ketosphinganine, D-threo-sphingosine and
C-sphinganine, and [1-
H]sphingosine
were synthesized according to Di Mari et al.(31) ,
Nimkar et al.(32) , Sarmientos et
al.(33) , and Cooke et al.(34) ,
respectively.
The [3-H]serine (30 mCi/mmol),
[14-
H]palmitate (54.4 Ci/mmol), and
[2-
C]ethanolamine-HCl (54.4 mCi/mmol) were from
Amersham Corp. The
-fluoro-L-alanine (
-F-alanine)
was donated by Merck (Rahway, NJ); FB
was purchased from
Sigma or from the Division of Food Sciences and Technology, Council for
Scientific and Industrial Research (Pretoria, South Africa).
In
experiments to evaluate the turnover of labeled sphingolipids, cells
were incubated with 0.1 mCi of [H]serine/ml
medium for 3 days, then the medium was removed, the cells were washed
twice with ice-cold PBS, and incubated in new DMEM with 10% FBS (or
HEPES-buffered DMEM for short term experiments).
To determine the sphingoid base 1-phosphate
mass, the cell lipids were extracted by the one-phase method described
for total sphingolipids and cleavage products (above), but
C-sphinganine was included as an internal standard. The
dried fraction was resuspended in 40 µl of methanol, followed by
0.1 ml of 0.1 M Tris, pH 8.8, 0.2 ml of 0.2 M glycine
buffer pH 9.0, 25 µl of H
0, 25 µl of 100
mM MgCl
, briefly sonicated(39) , and 25
units of alkaline phosphatase (from bovine intestinal mucosa (Sigma), a
nonspecific phosphatase that has been shown to hydrolyze phosphorylated
farnesylated derivatives)(38) , was added (pilot experiments
showed that approximately 70-80% of a sphingosine 1-phosphate
standard was cleaved to sphingosine under these conditions) (data not
shown). The tubes were capped under nitrogen and incubated at room
temperature for 6-8 h, then the long-chain bases were extracted
and quantitated by HPLC as described above.
To resolve individual lipid classes, aliquots of the lipid extracts were applied to Silica Gel 60 plates (Merck) and the chromatograms developed as follows: ceramide (diethyl ether/methanol, 99:1 v/v); long-chain bases (chloroform/methanol, 2 N ammonium hydroxide, 40:10:1.5 v/v); phosphatidylethanolamine and sphingoid bases (chloroform/methanol, 28% ammonium hydroxide, 13:7:1 v/v); complex sphingolipids, specifically gangliosides (chloroform/methanol, 0.22% calcium chloride, 60:35:8 v/v)(40) ; long-chain base cleavage products (hexane/diethyl ether/acetic acid, 70:30:1 v/v). Spots were generally detected by iodine; ninhydrin was used to visualize free long-chain bases. For autoradiography, TLC plates were sprayed with Fluoro-Hance (Research Products Intl., Mount Prospect, IL) and exposed to film for 5-7 days. In some experiments, radiolabeled lipids were detected on a Bioscan System 200 Imaging Scanner. Following identification with authentic standards, the radiolabeled lipids were scraped from the plate into scintillation vials, 0.2 ml of water and 4 ml of scintillation mixture were added, and the samples were counted for 10 min. Measurements are normalized for protein and expressed as disintegrations/min per mg protein, unless otherwise stated in the text. Protein was determined by the method of Lowry and co-workers (41) using BSA as standard. Phospholipid mass was determined by the measurement of inorganic phosphate content(42) .
Fig.1illustrates the changes in sphingosine and sphinganine mass that occurred when conditioned culture medium was removed from J774.A1 cells and fresh DMEM was added. Free sphingosine increased 6.4-fold within 15 min and reached a maximum (approximately 300 pmol/mg protein or an 8-fold increase) by 30 min. Sphinganine mass also rose, increasing approximately 10-fold in 15-30 min. The sum of these two long-chain bases was approximately 0.5 nmol/mg protein. This time course and the fold changes varied somewhat among experiments, but the maxima always occurred within an hour after changing the medium. Long-chain base levels typically decreased to about half of the maximum by 120 min (Fig.1) and returned to the initial level found in conditioned medium within 4 h (not shown).
Figure 1: Free sphingoid base mass after removal of conditioned culture medium. J774 cells were cultured for 3 days, then the conditioned culture medium was removed and replaced with fresh medium (without FBS) for various times. Sphingosine (open circles) and sphinganine (filled circles) were analyzed as described under ``Experimental Procedures.'' Results are the mean ± range (pmol/mg protein) from duplicate samples of a representative experiment.
Elevations in free sphingosine and sphinganine could arise from de novo synthesis and (or) turnover of sphingolipids. To
discriminate between these two possibilities, radiolabeled precursors
of long-chain base biosynthesis ([H]serine and
[
H]palmitate) were included in the fresh culture
medium and the incorporation of radiolabel into long-chain bases was
determined. [
H]Serine (Fig.2A)
was rapidly incorporated into free sphinganine, with the greatest
incorporation found at 15 and 30 min, a reduction to about half this
amount by 1 h, and little label in either free long-chain base after 2
h. This pattern of labeling resembled the time-dependent changes in
sphinganine mass (Fig.1). Based on the specific activity of the
added [
H]serine (30 mCi/mmol), the mass of
[
H]sphinganine present at 15 and 30 min would be
200 pmol/mg cell protein, and greater amounts could be present if
the [
H]serine is diluted with endogenous,
unlabeled serine. This amount was very close to the elevation in
sphinganine mass (200 pmol/mg protein) (Fig.1). Sphinganine
mass and labeling differed only at 2 h, which might be caused by
depletion of the labeled precursors (35) and/or to dilution of
the [
H]serine (see below). Sphinganine synthesis
from [
H]palmitate also increased (Fig.2B), but based on the specific activity of the
starting [
H]palmitate, only 1 pmol of
[
H]sphinganine/mg cell protein was produced,
which is less than 1% of the sphinganine mass. This difference is
probably due to dilution of the [
H]palmitate with
unlabeled, endogenous palmitate.
Figure 2:
[H]Serine and
[
H]palmitate incorporation into free sphingosine
and sphinganine. J774 cells were cultured for 3 days, then the medium
was removed and replaced with 1 ml of fresh DMEM containing 0.1 mCi
[
H]serine (A) or 1 µM palmitate spiked with 10 µCi of
[
H]palmitate (B).
[
H]Sphingosine (open circles) and
sphinganine (closed circles) were extracted, separated by TLC,
and quantitated by scintillation counting. Results are the mean
± S.D. (dpm/mg protein; n = 3) for A,
and mean ± half range (dpm/mg protein; n = 2)
for B.
There was little incorporation of
[H]serine and [
H]palmitate
into sphingosine (Fig.2, A and B, open
circles). Thus, the labeling time courses and changes in mass
indicate that the burst of sphinganine is due to increased de novo synthesis; sphingosine, however, arises from another source. These
findings are consistent with the pathway for sphingosine synthesis,
wherein the addition of the 4-trans double bond to sphinganine
occurs after N-acylation(35, 48, 60) .
Figure 3:
Effect of -F-alanine inhibition of de novo sphingolipid biosynthesis on sphingosine (A)
and sphinganine (B) masses following change to new medium.
J774 cells were cultured for 3 days, then
-F-Alanine (0
mM, open circles; 0.1 mM, filled
squares; 1.0 mM, filled triangles) was added 20
min prior to the removal of the conditioned medium and addition of new
DMEM containing
-F-alanine at the same concentrations. The
long-chain bases were quantitated after incubation for the indicated
times. Results are the average of duplicate samples ± half range
(pmol/mg protein).
Figure 4:
Serine stimulation of de novo sphingolipid synthesis. J774 cells were cultured for 3 days, then
changed to the indicated media, incubated for 15 min, and analyzed for
long-chain base mass. Results are expressed as the mean ± S.D.
(pmol/10 cells/dish, n = 3). Symbols:
sphingosine, solid bars; sphinganine, hatched bars.
The incubation media were as follows: NM, new HEPES-buffered
DMEM; CM, conditioned medium taken from the same or similarly
treated cells (both give the same result); CM + Ser, the
same conditioned medium supplemented with 0.4 mML-serine; PBS, 10 mM HEPES-buffered
phosphate-buffered saline; PBS + Ser, PBS supplemented
with 0.4 mML-serine.
Figure 5: Cellular amounts of serine and other amino acids in cells incubated in new medium. J774 cells were incubated in fresh DMEM (containing 10% FBS) for the given times, washed twice to remove the medium, and extracted for analyses of amino acids of the aqueous phase of the lipid extract as described under ``Experimental Procedures.'' The range for the data are within 10% of the mean for all measurements.
When cells were changed to PBS, the increase in sphingosine was comparable to new medium (Fig.4). Sphinganine increased 6-fold, which is 49% of the stimulation by new medium. Addition of 0.4 mM serine to PBS increased sphinganine by 40-fold over conditioned medium (6-fold over PBS alone) and had no effect on sphingosine. These results indicate that addition of exogenous serine enhances the sphinganine burst but is not necessary for some increase in sphinganine, presumably because cells can produce this amino acid precursor, and may release some sphinganine by hydrolysis of sphinganine-containing sphingolipids.
Figure 6: Chloroquine (A) and ammonium chloride (B) suppression of sphingoid base mass levels 1 h after change to new culture medium. Conditioned culture medium was removed, and cells were incubated in fresh DMEM containing either chloroquine or ammonium chloride for 1 h, after which sphingosine (open circles) and sphinganine (filled circles) were extracted and quantitated. Values represent the mean of triplicate determinations and the S.D. lie within 10% of the mean for all values except the 5 µM chloroquine data where the S.D. for sphingosine are ± 19% of the mean and sphinganine ± 16% of the mean. The sphingosine and sphinganine mass in control cells were 252 ± 23 and 164 ± 5.6 pmol/mg cell protein (mean ± S.D.), respectively, for the chloroquine experiment. In control cells for the ammonium chloride experiment, the sphingosine mass was 377 ± 59 pmol/mg protein, and sphinganine was 381 ± 44 pmol/mg protein.
Figure 7:
Changes in sphingoid base composition of
ceramide and total sphingolipids following the change to new culture
medium. After incubation for various times in new DMEM, J774 cells were
extracted and the sphingosine (open circles) and sphinganine (filled circles) content of ceramides (A) and total
sphingolipids (C) determined as described under
``Experimental Procedures.'' The incorporation of
[H]serine (0.1 mCi/ml, added at t
) was determined by acid hydrolysis of the
ceramide (B) and total sphingolipids (D) to sphingoid
bases, which were separated by TLC and quantitated by scintillation
counting. Results are the mean ± S.D. (dpm/mg protein) of
triplicate samples.
The amount of ceramide increased 3-fold (Fig.7A, closed circles) within 30 min and remained elevated at approximately 200 pmol/mg protein for 120 min. Ammonium chloride (10 mM) completely abrogated the increases in ceramide, but not in dihydroceramide (data not shown). The lack of an observable decrease in the mass of complex sphingolipids (Fig.7C, open circles) is probably due to the small amounts of ceramide and free sphingosine that are produced (400-500 pmol) compared to the total sphingolipid mass (>6 nmol).
Analyses of the labeled long-chain bases (Fig.7, B and D) established that over this time course,
[H]serine was incorporated almost exclusively
into the sphinganine backbone of dihydroceramide (Fig.7B, closed circles), with little
conversion to ceramide (Fig.7B, open
circles), which further substantiates the hypothesis that ceramide
is derived mainly from the turnover of unlabeled sphingolipids rather
than from the conversion of dihydroceramide to ceramide. The labeling
of sphinganine and sphingosine in total sphingolipids resembles a
precursor-product relationship (Fig.7D): the
[
H]sphinganine in complex sphingolipids rose
steadily in the first hour and thereafter leveled off, whereas
[
H]sphingosine increased more gradually and did
not equal [
H]sphinganine until 2-4 h (Fig.7D, openversusclosed
circles). Only a portion (23% after 30 min and 11% after 4 h) of
this label was in dihydroceramide per se, with the remainder
in complex sphingolipids. These data reveal that a substantial amount
of dihydroceramide can be incorporated into complex sphingolipids and
raise the possibility that the 4,5-trans-double bond might be
added to complex (dihydro)sphingolipids, as well as to
dihydroceramides(60) .
Figure 8:
Turnover of
[H]serine-labeled sphingolipids. J774 cells were
plated at a density of 5
10
cells/2 ml in 60-mm
dishes in DMEM containing 10% FBS and 0.1 mCi/ml
[
H]serine. After 3 days, the conditioned medium
was removed, and the cells were washed with PBS and incubated in fresh
DMEM with or without 2 or 10 mM ammonium chloride
(NH
Cl). After 1 h, the lipids were extracted and analyzed
as described under ``Experimental Procedures.'' Free
sphingosine (Free So) was determined by HPLC, and the
long-chain bases of sphingomyelin (SM), lactosylceramide (LacCer), and ceramide (Cer) were quantitated after
acid hydrolysis as described in the text. Results are expressed as
dpm/µg protein ± S.D. (n =
3).
During the first 20 min, label appeared in
3-ketosphinganine (Fig.9, triangles), sphinganine (circles), and N-acyl-sphinganines (squares). The chase reduced, but did not abolish, the
labeling of sphinganine and N-acyl-sphinganine; nonetheless,
there was a significant reduction in N-acyl[H]sphinganine after addition of
FB
and an equivalent increase in
[
H]sphinganine and
3-keto-[
H]sphinganine compared to both the
control and the 40-min time point. Thus, at least in this system,
sphinganine (and possibly 3-ketosphinganine) can be acylated as well as
released from newly made dihydroceramides.
Figure 9:
Incorporation of
[H]serine into newly synthesized long-chain bases
and N-acyl-sphinganine (dihydroceramide) in vitro. A
cell membrane fraction was incubated with the precursors for N-acyl-sphinganine as described in the text. After 20 min, 1
mM cold serine, 1 mM
-F-alanine, and 0.1 mM stearoyl-CoA were added (indicated as the Chase); and
after an additional 20 min (at FB
), 25
µM FB
was added to the groups designated by
the striped line. At each time, the amount of radiolabel in
sphinganine (closed circles), dihydroceramide (open
squares), and 3-ketosphinganine (closed triangles) were
determined. Results are expressed as the mean ± S.D. (pmol/mg
protein, n = 3), and represents one of two experiments
that gave this profile.
Figure 10: Determination of phosphorylated sphingoid bases. Free sphingosine (filled circles) and sphinganine (open circles) were analyzed directly by HPLC, and the 1-phosphates were determined by incubation of the lipids with 20 units of alkaline phosphatase for 6-8 h, followed by analysis of sphingosine (filled squares) and sphinganine (open squares) by HPLC. Results are expressed as pmol/mg cell protein (mean ± S.D., n = 4).
The data in Fig.10suggest that newly made
sphinganine, as well as sphingosine, are accessible to both ceramide
synthase and sphingosine kinase. If so, inhibition of acylation by
FB should divert more of the long-chain base toward
phosphorylation. Incubation of the cells with 50 µM FB
caused sphinganine to increase from 6.3 ±
1.9 pmol/dish at time 0 to 169 ± 9.9 pmol/dish after 30 min
(compared to 77 ± 6 pmol/dish for cells that were not treated
with FB
). A comparable increase was found in sphinganine
1-phosphate (Fig.11), with the greatest effect of the FB
seen at 120 min. In contrast, FB
had little effect on
the amounts of sphingosine (i.e. 88 ± 7 versus 81 ± 15 pmol/dish at 30 min with and without
FB
, respectively) or on sphingosine 1-phosphate (Fig.11). Therefore, FB
had little effect on
sphingosine accumulation or on its phosphorylation.
Figure 11:
Long-chain base 1-phosphate
concentrations in the presence of FB. At the time of
changing the cells to fresh medium, 50 µM FB
was added and sphingosine 1-phosphate (open bar) and
sphinganine 1-phosphate (solid bar) were determined as
described under ``Experimental Procedures'' and the legend
for Fig.10. Results are expressed as pmol/mg cell protein (mean
± S.D., n = 4).
When J774 cells were labeled with 10 µCi/ml
[H]serine for 1 or 12 h in the presence and
absence of 50 µM FB
(Table1), FB
reduced the amount of [
H]sphingosine in
ceramide, sphingomyelin, and total sphingolipids, but did not alter the
incorporation of radiolabel into the total lipid extract, PC, or PS. In
contrast, FB
increased the amount of radiolabel in PE by 48
and 31% after 1 and 12 h, respectively. Incubation of J774A.1 cells
with 50 µM FB
also did not alter the mass of
PE significantly (36.3 ± 3.9 nmol/mg protein for
FB
-treated and 40.5 ± 0.9 nmol/mg protein for
control cells, or approximately 25% of the total cellular phospholipid,
at 12 h), whereas the sphingomyelin mass decreased by 46% (from 18.4
± 1.0 nmol/mg protein to 9.9 ± 1.2 nmol/mg protein).
These findings are consistent with FB
diverting newly made
[
H]sphinganine 1-phosphate to
[
H]ethanolamine phosphate for use in
[
H]PE synthesis, and raises the possibility that
sphinganine serves not only as an intermediate of sphingolipid
biosynthesis, but also as a source of ethanolamine phosphate in some
cells. A similar observation was made, but not explained, in studies of
the effects of FB
on hepatocytes(24) .
Ethanolamine phosphate synthesized via the long-chain base
phosphates should also compete with the incorporation of exogenously
added [C]ethanolamine into PE. When J774 cells
were incubated with 0.25 µCi of
[
C]ethanolamine in the fresh culture medium,
FB
reduced the amount of label incorporated into
[
C]PE (Fig.12). The inhibition (30% at
12 h) was comparable to the increase in incorporation of
[
H]serine into PE (31%, Table1).
Figure 12:
Effect of FB on the
incorporation of [
C]ethanolamine into PE. Cells
were cultured for 3 days, and new medium containing
[
C]ethanolamine (0.25 µCi) with (closed
circles) or without (open circles) 50 µM FB
was added and the amount of radiolabel in PE
determined. Results are expressed as dpm/nmol PE/mg cell protein (mean
± S.D., n = 3).
Since
long-chain bases (58) and the 1-phosphates (59) have
been reported to activate a PE-specific phospholipase D, FB might induce turnover and reutilization of the
ethanolamine-derived headgroup. This possibility was eliminated
because, when J774 cells were cultured for 48 h in medium containing
0.25 µCi/ml [
C]ethanolamine and subsequently
changed to new DMEM containing 2 mM cold ethanolamine with or
without FB
, the rate of release of label from
[
C]phosphatidylethanolamine was 4.2 ± 1.0
pmol/min/mg protein for both groups (data not shown).
Utilization of long-chain bases for PE biosynthesis was demonstrated
directly by synthesizing [1-H]sphingosine and
following the fate of the radiolabel upon incubation with the cells (Table2). After 3 h, about half of the radiolabel was recovered
in the aqueous phase (Table2) which most likely represents
formation of [
H]ethanolamine. Consistent with
this interpretation, about half of the label in the organic phase was
in PE, with the remainder distributed mostly among free sphingosine,
ceramide, and sphingomyelin (Table2).
When the cells were
also treated with 50 µM FB, there was a
reduction in the amount of label in sphingomyelin (6.7-fold) and
ceramide (1.4-fold) due to inhibition of
[1-
H]sphingosine acylation (Table2). The
disintegrations/minute in the aqueous phase increased 30-40%, as
expected for increased phosphorylation (as shown above) and cleavage of
the [1-
H]sphingosine; however, the
disintegrations/minute in PE were somewhat lower than in the cells that
were not treated with FB
. This seeming contradiction was
explained by analysis of the mass of the long-chain bases in the cells.
When the mass of unlabeled sphinganine and sphingosine is used to
estimate the specific activity of the
[
H]long-chain bases (Table2), the specific
activity is 45% lower for the FB
-treated cells than for the
controls. Therefore, the mass of PE that would correspond to the
disintegrations/minute (Table2) would be 580 pmol/mg protein in
FB
-treated cells and 400 pmol/mg protein in control cells.
These calculations are approximations because the specific activities
of all of the relevant intermediates are not known; nonetheless, this
increase in PE synthesis from
H-long-chain bases in the
FB
-treated cells is almost identical to the findings from
[
H]serine labeling and the inhibition of
[
C]ethanolamine incorporation (i.e. 30%, as described above).
The catabolism of exogenous
[1-H]sphingosine and reutilization for PE
synthesis even in cells that were not treated with FB
was
examined further (Fig.13). At the earliest time point examined
(15 min), over half of the cell-associated radiolabel was found in the
aqueous phase of the lipid extract. Sphingosine, ceramide,
sphingomyelin, and PE accounted for most of the radiolabel in the
organic phase. The time course for the disintegrations/minute in
sphingosine and ceramide versus PE and sphingomyelin suggested
a precursor-product relationship.
Figure 13:
[1-H]Sphingosine
uptake and metabolism. [1-
H] Sphingosine was
added as a 1:1 (mol/mol) sphingosine-BSA complex to a final
concentration of 1 µM at time 0 in fresh culture medium
(DMEM). Radioactivity associated with the total cell extract, the
aqueous phase recovered from the two-phase cellular lipid extraction,
and the individual lipids were determined as described under
``Experimental Procedures.'' Results are expressed as the
mean ± S.D. of triplicate samples.
The findings of this study are summarized by the scheme shown in Fig.14. Replacement of conditioned with fresh culture medium on J774A.1 cells induces a rapid burst of long-chain bases from at least two sources: sphinganine and its derivatives (sphinganine 1-phosphate, N-acyl-sphinganines, etc.) that are newly synthesized, and sphingosine and its derivatives (sphingosine 1-phosphate and N-acyl-sphingosines) that are produced by the turnover of existing complex sphingolipids. Since we first described this change with J774A.1 cells(27, 73, 74, 75) , we have noted similar bursts in Swiss 3T3 cells(28) , primary cultures of rat hepatocytes, and mouse peritoneal macrophages (data not shown), and Lavie et al.(29) have made similar observations with NIH-3T3 cells, A431 cells, and NG108-15 cells. While the biological significance (if any) of this apparently common phenomenon is unknown, it has provided a useful model system for dissecting the factors that influence the appearance and fate of free long-chain bases in cells.
Figure 14:
An overview of endogenous long-chain base
metabolism in J774A.1 macrophages. Shown are the interrelationships
found by this study. The abbreviations represent 3-ketosphinganine (KA), sphinganine (Sa), N-acyl-sphinganine
or dihydroceramide (N-Acyl-Sa), ceramide (N-Acyl-So),
sphingosine- and sphinganine 1-phosphates (Sa-P and So-P), PE, -fluoro-alanine (
F-ala), and
FB
. This figure illustrates the elements of this pathway
that are most relevant to this article, but does not include all of the
intracellular reactions of sphingolipid metabolism (such as the
turnover of sphingolipids in the plasma membrane), nor does it show all
elements of their topologic distribution (for example, the first step
of glycolipid synthesis occurs on the cytosolic leaflet of the
Golgi).
The 4-trans double bond of sphingosine is
inserted subsequent to the formation of N-acyl-sphinganine
(dihydroceramide) during sphingolipid biosynthesis de novo(35, 48, 60) ; thus, free sphinganine
appears as an intermediate of the de novo pathway, with
sphingosine arising from hydrolysis of complex sphingolipids. The
findings of this study confirm this pathway because the amounts of
sphinganine (but not sphingosine) were affected by -F-alanine (as
an inhibitor of serine palmitoyltransferase) (49) as well as by
varying the amounts of serine. The strong dependence of long-chain base
biosynthesis on the availability of this precursor has been seen in a
variety of cell types (mouse LM cells, hepatocytes, and J774
macrophages) (50, 51, this study), which suggests that this may be one
of the factors that regulates this pathway. If so, sphingolipid
metabolism may be affected by hormonal and nutritional factors that
govern amino acid metabolism. There have been no reports of hormones
affecting de novo long-chain base synthesis; however, a recent
study noted that insulin augmented the accumulation of sphinganine in
FB
-treated Swiss 3T3 cells(28) .
N-Acylation appears to account for the majority of sphinganine metabolism, particularly after the initial burst has dissipated. Substantial amounts of sphinganine appeared first in dihydroceramides and more complex sphingolipids, and sphingosine later became the major long-chain base of the complex sphingolipids. A similar observation has been made with LM cells(35) . Apparently, the 4,5-double bond is not required for addition of the headgroups, yet ultimately appears in the backbones of complex sphingolipids. Evidence for an enzyme activity that adds a double bond to dihydroceramides has been reported(60) , but our findings raise the possibility that as-yet-uncharacterized enzyme(s) may act also on more complex substrates. Alternatively, newly made (sphinganine-containing) sphingolipids may be turned over rapidly (61) and the double bond added before the (dihydro)ceramide backbone is reutilized. Hannun and co-workers (12, 14) have speculated that formation of ceramides as intermediates of the de novo biosynthetic pathway could be deleterious because they can affect cell growth and induce apoptosis. Therefore, introduction of the 4,5-double bond after addition of headgroups to dihydroceramides would avoid this potential danger.
In J774A1 cells, free sphingosine is derived primarily from hydrolysis of complex sphingolipids in an acidic compartment(s), perhaps reflecting the endosomal and/or lysosomal turnover of sphingolipids. A source of the sphingosine appears to be sphingomyelin, based on the labeling studies. However, the amounts of free sphingosine are only a small percentage of the cellular sphingolipid mass; therefore, turnover of other sphingolipids could also contribute.
We estimate that
approximately 30% of endogenous sphingosine generated during the burst
period is reincorporated into sphingolipids; this relatively low
reutilization agrees well with the rapid (<15 min) generation of
water-soluble metabolites and the substantial utilization of
[1-H]sphingosine for phosphatidylethanolamine
biosynthesis. The difference in sphingosine and sphinganine metabolism
may be related to the location of the enzymes involved in acylation and
phosphorylation. Along with serine palmitoyltransferase and the
NADPH-dependent 3-ketosphinganine reductase(62, 63) , N-acyl-sphingosine (sphinganine) transferase is localized to
the external leaflet of the ER; sphingosine kinase, however, has been
found in cytosol and localized to microsomal
membranes(56, 57, 64, 65) , and
sphingosine 1-phosphate lyase is associated mainly with the endoplasmic
reticulum(66) . Therefore, sphingosine that is liberated in
lysosomes (or other acidic compartments), and perhaps also in the
plasma membrane(67) , must move to other intracellular
locations to be metabolized further, providing opportunities for it to
be phosphorylated and cleaved.
Our findings demonstrate that the
burst, alone or in combination with inhibition of N-acylation
of sphingoid bases by FB, results in an augmented flux of
long-chain bases toward the 1-phosphates and cleavage, which in turn
apparently affects the cellular ethanolamine phosphate pool used for PE
biosynthesis via the CDP-ethanolamine pathway. Based on the changes
that FB
treatment induced in PE labeling (and on the
assumption that the other PE biosynthetic pathways were not affected),
sphingolipid degradation contributed approximately 0.1-1 nmol of
ethanolamine/hour to total PE synthesis. This rate is feasible
considering the amounts of long-chain bases that are formed by de
novo synthesis and shuttled toward phosphorylation and cleavage.
Therefore, although the quantitative significance of this pathway is
still unclear, this study indicates that sphinganine could be a
significant source of ethanolamine in some cell types.
The formation of such large amounts of the long-chain base 1-phosphates is also intriguing because sphingosine 1-phosphate has potent effects on cells (reviewed in (16, 17, 18) ). Spiegel and co-workers have shown that sphingosine 1-phosphate stimulates proliferation of quiescent Swiss 3T3 fibroblasts(68) , and have suggested that this may be mediated through several pathways, including release of intracellular calcium(25) , increases in phosphatidic acid levels(58) , and modulation of AP1(26) . They have also found that sphingosine 1-phosphate (as well as sphingosine) increases in Swiss 3T3 cells treated with platelet-derived growth factor(16) . Studies in permeabilized cells have implicated sphingosine 1-phosphate in the release of calcium from intracellular stores(15) , and Ghosh et al.(17) have reported that the conversion of sphingosine to sphingosine 1-phosphate by the sphingosine kinase associated with the endoplasmic reticulum activates release of stored calcium.
It is
tempting to speculate that these changes in long-chain bases and their
derivatives (both the 1-phosphates and N-acyl-derivatives)
affect the behavior of J774 cells. The increases in sphingosine and
sphingosine 1-phosphate after changing the medium are comparable to the
amounts seen in platelet-derived growth factor-treated Swiss 3T3 cells (16) and stimulation of Swiss 3T3 cell growth by
FB(28) . In other cell systems, sphingosine and
sphinganine are growth inhibitory and cytotoxic at these levels (69) , and ceramides have been found to have diverse effects,
from growth stimulation (70) to induction of
apoptosis(71) . In recent studies(72, 76) , we
have found that J774 cells undergo changes in phorbol dibutyrate
binding and a number of other cellular responses that would be
predicted to occur when the amounts of sphingosine increase after
changing the medium. Thus, the burst may play a role in the generally
known (but rarely mentioned) anomalies in cell behavior after changing
the medium that necessitate that many types of cells be given several
hours to ``calm down'' before beginning an experiment.
Two findings of this study should be borne in mind by
investigators who are analyzing the role of free long-chain bases and
their derivatives (N-acyl-compounds, 1-phosphates, and
probably others) as mediators of the action of hormones, growth and
differentiation factors, and cytokines. First, it appears that many
cells undergo large increases in these compounds after changing the
culture medium; therefore, care must be taken to distinguish between
this perturbation and the response that is of interest. Second, many
factors differentially alter the metabolism of sphinganine versus sphingosine, ceramide versus dihydroceramide, etc.
Therefore, analyses of these compounds by methods that do not
distinguish between the presence of the 4,5-trans double bond
will not determine whether an agonist is producing the presumed
bioactive product: for example, the use of diglyceride kinase to
analyze ceramides as ceramide-1-[P]phosphate
does not discriminate between ceramide released from existing
sphingolipids and dihydroceramide produced via stimulated de novo biosynthesis. While these questions are complex, the approaches
described in this article should be helpful in distinguishing among the
possibilities.