©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Sphingolipid Biosynthesis de Novo by Rat Hepatocytes in Culture
CERAMIDE AND SPHINGOMYELIN ARE ASSOCIATED WITH, BUT NOT REQUIRED FOR, VERY LOW DENSITY LIPOPROTEIN SECRETION (*)

(Received for publication, October 31, 1994; and in revised form, March 27, 1995 )

Alfred H. MerrillJr. (1)(§) Susanne Lingrell (2) Elaine Wang (1) Mariana Nikolova-Karakashian (1) Teresa R. Vales (1) Dennis E. Vance (2)

From the  (1) Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322-3050 and the (2) Lipid and Lipoprotein Research Group and Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada T6G 2S2

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Sphingolipids are constituents of liver and lipoproteins, but relatively little is known about their synthesis and secretion. Incubation of rat hepatocytes with [^14C]- or [^3H]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(1), 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.


INTRODUCTION

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 [^14C]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 [^14C]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.


EXPERIMENTAL PROCEDURES

Materials

Media for cell culture were purchased from Life Technologies, Inc.; collagenase (Type IV), fatty acid-free bovine serum albumin, and other tissue culture reagents were obtained from Sigma. The [U-^14C]serine (55 mCi/mmol) and [^3H]serine (10-30 Ci/mmol) were from Amersham (United Kingdom or Arlington Heights, IL); [U-^14C]serine (135 mCi/mmol) was also obtained from ICN Radiochemicals (Irvine, CA) and diluted to ca 50 mCi/mmol with unlabeled serine. [^3H]Sphingosine was a gift from DuPont NEN and was purified by TLC before use (Merrill and Wang, 1992). Sphingomyelin, ceramide, fatty acids, fatty acyl-CoA, and the other lipid standards were purchased from Sigma. Fumonisin B(1) was obtained from the suppliers described previously (Wang et al., 1991).

Cells

Hepatocytes were prepared by the method of Yao and Vance(1988) using livers from male Sprague-Dawley rats (45-200 g) fed ad libitum a standard chow diet (Purina Rat Chow, Ralston Purina, St. Louis, MO) or a formulation to induce choline deficiency as described previously (Yao and Vance, 1988). Cell viabilities were >90% as assessed by the exclusion of 0.2% trypan blue.

Incubation of Cells with Radiolabeled Serine

The incubations were conducted essentially as described previously (Messmer et al., 1989; Yao and Vance, 1988). This involved culturing the hepatocytes (2.5-4.3 mg of protein, determined by the Lowry method with BSA^1(^1) as the standard) on collagen-treated dishes (30 mm) for 4 h, then the medium was removed, the cells washed, and new medium containing radiolabeled serine (0.4 mM final concentration at 25-50 mCi/mmol) and other factors were added. When fatty acids were added to the cells, they were first prepared as complexes with fatty acid-free BSA (Sigma) by mixing the fatty acid in ethanol with BSA in phosphate-buffered saline (PBS), followed by dialysis overnight against PBS. During the incubation, the cells were maintained at 37 °C and a humidified atmosphere of 5% CO(2) in a tissue culture incubator.

Lipid Analyses

The ^14C-labeled long-chain bases were analyzed as described previously (Merrill and Wang, 1986; Messmer et al., 1989). The cells were scraped from the dishes, added to glass test tubes, and 25 µg of carrier sphingolipids (sphingomyelin, ceramide, and sphingosine) were added. After extracting the lipids, aliquots were acid hydrolyzed to determine the total sphingolipids by analysis of the radiolabel in sphingosine and sphinganine (Merrill and Wang, 1986), ceramide (after separation by silica gel TLC in diethyl ether:methanol, 99:1, v/v), and sphingomyelin (after TLC in CHCl(3):methanol:acetic acid:water, 56:30:4:2, v/v). The amount of radiolabel was corrected using an estimate for quenching by the silica gel that was obtained by adding a known amount of radiolabel to the chromatogram. Analyses of the radiolabel in long-chain base backbones of ceramide and sphingomyelin according to Merrill and Wang(1986) revealed that >80% of the disintegrations/min could be accounted for in sphingosine plus sphinganine; therefore, the disintegrations/min could be compared directly with the total sphingolipid analyses (which were based on analysis of disintegrations/min in the long-chain bases).

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 [^3H]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 [^3H]ceramide samples were analyzed by HPLC using a Waters C(18)-µBondapak column and acetonitrile:ethanol (60:40, v/v) as the mobile phase. The radiolabel was detected using a beta-Ram flow-through monitor.

To produce [^3H]ceramides as standards for the HPLC analyses, various fatty acyl-CoAs were incubated with [^3H]sphingosine as described by Merrill and Wang(1992), and the products were analyzed as described above (the retention times of the various N-acyl-[^3H]sphingosines are given in Fig. 5 ).


Figure 5: Analyses of [^3H]ceramides in cells (middle panel) and medium (lower panel) after incubation of hepatocytes with [^3H]serine. Hepatocytes (3-4 mg/dish) were incubated overnight with 100 µCi of [^3H]serine, then the [^3H]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 [^3H]ceramides were separated by reverse-phase HPLC (Waters C(18)-µBondapak column) with acetonitrile:methanol (60:40, v/v) as the eluting solvent) and quantitated with an in-line radioactivity detector. Standard [^3H]ceramides were prepared by incubating [^3H]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.



Lipoprotein Analyses

To determine the radiolabel in lipoproteins, the culture medium was removed, centrifuged for several minutes at 8,000 times g to remove dislodged cells, then subjected to centrifugation in a sucrose gradient (Yao and Vance, 1988) or sequential centrifugations at different densities (Havel et al., 1955). Very low density (VLDL), low density (LDL), and high density (HDL) lipoproteins were isolated from rat plasma by the same method using the density ranges d < 1.018 g/ml for VLDL, 1.018 < d < 1.063 for LDL, and 1.063 < d < 1.18 for HDL. The lipids were extracted, separated by TLC, and analyzed for radiolabel (Merrill and Wang, 1986) or mass by HPLC (Merrill et al., 1988b).

To determine the effect of fumonisin B(1) 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(1). 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).


RESULTS

An earlier study (Messmer et al., 1989) showed that hepatocytes incorporate [^14C]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 [^14C]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 [^14C]serine. Hepatocytes (approximately 2.7 mg of protein/culture dish) were incubated with 20 µCi of [^14C]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 [^14C]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 [^14C]serine, the cellular level of [^14C]ceramide is at least 0.3 nmol/mg of protein after 4 h of labeling. A more exact estimate of the amount of [^14C]ceramide is precluded by the dilution of the [^14C]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 [^14C]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 [^14C]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 [^14C]serine into ceramide, sphingomyelin, and total sphingolipids.

Sphingolipid Biosynthesis de Novo and Secretion with Lipoproteins

The majority of the radiolabeled sphingolipids was recovered with the cells; however, from 1 to 5% appeared in the culture medium (Fig. 2). The predominant sphingolipid in the medium was [^14C]ceramide (Fig. 2B).


Figure 2: Time course of sphingolipid labeling from [^14C]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 [^14C]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 [^3H]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 [^3H]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 times 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 [^14C]serine (Fig. 4, left panel) and [^3H]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 [^14C]serine (left panel) or 0.5 mCi of [^3H]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 times 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 [^14C]serine and a single sample incubated with [^3H]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.

Ceramide Is a Significant Component of VLDL in Vivo

As far as we are aware, ceramide has not been previously reported to be a constituent of lipoproteins; therefore, we analyzed the ceramide amounts in lipoproteins isolated from rat plasma to determine if the release of labeled ceramide from hepatocytes is an artifact of cell culture. As shown in Table I, all of the lipoprotein fractions contain ceramide; however, it is the most predominant species (i.e. about 50% of the total sphingolipid) in the lipoproteins with d < 1.018 g/ml. Sphingomyelin accounted for most of the sphingolipid in LDL and HDL, which agrees with the higher percentages of sphingomyelin in the phospholipids of LDL (6.5 versus 49.7% phosphatidylcholine) and HDL (6.2 versus 59.1% phosphatidylcholine) compared to VLDL (4.2 versus 75.5% phosphatidylcholine) (Chapman, 1986). Small amounts of free sphingosine (Table I) were also detected in each lipoprotein fraction.

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.

Effects of Fatty Acids on Sphingolipid Synthesis and Secretion

Because the addition of palmitic acid to isolated hepatocytes has been shown to increase the incorporation of [^14C]serine into sphingolipids in short term studies (Messmer et al., 1989), this parameter was examined also for these studies of hepatocytes over longer time courses. Addition of palmitic acid as the BSA complex increased total sphingolipid and sphingomyelin labeling by about 2-fold when added at a palmitic acid:BSA ratio (mol:mol) of 2:1 (Fig. 6, left panel). Fatty acid-free BSA caused some stimulation (25%), and this was not seen if BSA was provided as the 1:1 complex with oleic acid. Stearic acid (at a 2:1 molar ratio with BSA) had about the same effect as BSA alone. This selectivity in the stimulation of long-chain base biosynthesis by fatty acids has been attributed to the substrate specificity of the first enzyme of sphingolipid biosynthesis (serine palmitoyltransferase) for saturated fatty acyl-CoAs of 16 ± 1 carbon atoms (Merrill et al., 1988a; Messmer et al., 1989).


Figure 6: Stimulation of sphingolipid biosynthesis from [^14C]serine by palmitic acid. Hepatocytes (approximately 2.5 mg/dish) were incubated with 20 µCi of [^14C]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 [^14C]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 [^14C]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.

Sphingolipid Biosynthesis Is Not Impaired in Choline-deficient Hepatocytes

Vance and co-workers (Yao and Vance, 1988, 1990; Vance, 1990) have shown that the biosynthesis of phosphatidylcholine is restricted by feeding rats diets deficient in choline and culturing the hepatocytes in medium free of choline and methionine and that this restricts lipoprotein secretion by hepatocytes. Because the headgroup of sphingomyelin is thought to be derived mainly from phosphatidylcholine (as reviewed in Merrill and Jones(1990)), the effects of choline deficiency on sphingolipid biosynthesis was determined (Table II). In both of the experiments that were conducted, the overall sphingolipid labeling was not affected significantly by incubation of the hepatocytes with or without choline and methionine (Table II).

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 [^14C]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 [^14C]serine. Hepatocytes (approximately 2.7 mg of protein/dish) were incubated with 20 µCi of [^14C]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(3):methanol:acetic acid:H(2)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.

Synthesis and Secretion of Sphingolipids and Other Lipids by Hepatocytes Treated with Fumonisin B(1)

A possible role for sphingomyelin in the secretion of lipoproteins by hepatocytes cannot be excluded because choline deficiency did not appear to affect sphingomyelin formation. To address this question by an alternative approach, hepatocytes were incubated with fumonisin B(1), an inhibitor of ceramide synthase (Wang et al., 1991). Fumonisin B(1) reduced overall sphingolipid biosynthesis by about 90% (Table III) and reduced the labeling of sphingomyelin by 84% and ceramide by 96%. There was no decrease in the amount of radiolabel in the cell-associated phosphatidylcholine or cholesterol.

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(1) 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(1), respectively; after 24 h, the amounts were 324 ± 48 versus 304 ± 34 in the absence and presence of 10 µM fumonisin B(1), respectively (n = 3). These results establish that sphingolipid biosynthesis can be inhibited substantially without having an effect on lipoprotein secretion by hepatocytes.


DISCUSSION

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 alpha1-acid glycoprotein and suppression of CYP2C11) and that sphingomyelin hydrolysis is stimulated by interleukin-1beta,^2(^2) 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 [^3H]- or [^14C]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 [^2H]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 [^14C]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-alpha 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(1) 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(1) 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 [^
14C]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.




   Table III: 0p4in


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)


FOOTNOTES

*
This work was supported by National Institutes of Health Grant GM46368 and a Pew Nutrition Scholars Fellowship (to A. H. M.) and funds from the Heart and Stroke Foundation of Alberta (to D. E. V.). This work was presented in part at the 1990 meeting of the Federation of the American Societies for Experimental Biology (Merrill, A. H., Jr., Lingrell, S., Wang, E., and Vance, D. E. (1990) FASEB J.4,779a). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement''in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Dept. of Biochemistry, 4113 Rollins Research Center, Emory University, Atlanta, GA 30322. Tel.: 404-727-5978; Fax: 404-727-3954.

(^1)
The abbreviations used are: BSA, bovine serum albumin; DMEM, Dulbecco's modified Eagle's medium; HDL, high density lipoproteins; LDL, low density lipoproteins; PBS, phosphate-buffered saline; TLC, thin-layer chromatography; VLDL, very low density lipoproteins. The nomenclature used in this paper generally conforms to the recommendations of IUPAC/IUB. The terms sphingosine and sphinganine have been used for the long-chain bases with and without the 4-trans-double bond without specification of the alkyl chain length.

(^2)
J. Chen, M. Nikolova-Karakashian, A. H. Merrill, Jr., and E. T. Morgan, submitted for publication.


ACKNOWLEDGEMENTS

We thank Dr. Patrick Tso for the providing the lymph used for preliminary analyses of the ceramide content of rat intestinal chylomicrons and Dr. Martin Houweling for the cells used in Fig. 5 .


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