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Elevation of ceramide in serum lipoproteins during acute phase response in humans and mice: role of serine–palmitoyl transferase
ABB Archives of Biochemistry and Biophysics 419 (2003) 120–128 www.elsevier.com/locate/yabbi
Elevation of ceramide in serum lipoproteins during acute phase response in humans and mice: role of serine–palmitoyl transferase Sandy Lightle,a Raina Tosheva,b Amy Lee,a Jennie Queen-Baker,a,b,c Boris Boyanovsky,a Steve Shedlofsky,b,c and Mariana Nikolova-Karakashiana,* a Department of Physiology, University of Kentucky Lexington, KY 40536, USA Department of Medicine, VA Medical Center, University of Kentucky Lexington, KY 40536, USA General Clinical Research Center, College of Medicine, University of Kentucky Lexington, KY 40536, USA b
c
Received 27 January 2003, and in revised form 15 August 2003
Abstract Recent studies have indicated that ceramide generated in the liver is secreted into the bloodstream as component of very-lowdensity lipoproteins (VLDL) and low-density lipoproteins (LDL). This manuscript investigates the effect of host acute phase response to inflammation on lipoprotein ceramide levels. In humans, two different patterns of responses were found. One group of volunteers experienced transient increases in serum ceramide at 1.5 h after LPS administration. Second group showed prolonged increases that reached up to 10-fold above the basal level and continued for up to 24 h. Increases in ceramide were found only in VLDL and LDL particles. LPS administration induced similar increases in mice. These increases were accompanied by activation of secreted sphingomyelinase in serum and serine–palmitoyl transferase in liver. ASMase knockout mice retained LPS-induced increases in serum ceramide, thus suggesting that the elevation of VLDL and LDL ceramide content is attributed at least in part to activation of de novo synthesis of ceramide in the liver. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Atherosclerosis; LPS; Liver; Sphingomyelinase; Sphingosine
Ceramide, a sphingolipid second messenger molecule, has been extensively studied for its role in cellular response to stress and inflammation. Key inducers and mediators of host acute phase response (APR),1 such as bacterial lipopolysaccharide (LPS), Interleukin 1b (IL1b), and Tumor necrosis factor a (TNFa), induce early generation of ceramide in the liver [1–3]. In isolated hepatocyte cultures, the accumulation of ceramide occurs within 1 h of exposure to IL-1b and is caused by activation of a neutral sphingomyelinase (SMase), an
*
Corresponding author. Fax: 1-606-323-1070. E-mail address: [email protected] (M. Nikolova-Karakashian). 1 Abbreviations used: VLDL, very-low-density lipoproteins; LDL, low-density lipoproteins; LPS, lipopolysaccharide; IL-1b, interleukin 1b; TNFa, tumor necrosis factor a; SMase, sphingomyelinase; SPT, serine–palmitoyl transferase; SSMase, secreted SMase; LPDS, lipoprotein-deficient serum; LDLR, LDL receptor. 0003-9861/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2003.08.031
enzyme that metabolizes SM in cellular membranes to phosphorylcholine and ceramide. The latter appears to mediate the cytokine-induced expression of serum acute phase proteins, including a1 -acid glycoprotein, C-reactive protein, and serum amyloid A [1,4]. The agonistinduced activation of sphingomyelinase is often coupled to activation of ceramidase and sphingosine kinase that convert ceramide to other bioactive sphingolipids, sphingosine and sphingosine-phosphate, thus restoring the normal cellular levels of ceramide [5]. Studies in a hamster model of inflammation suggest that in addition to activation of sphingomyelinase, liver APR leads to increases in de novo synthesis of ceramide via activation of serine–palmitoyl transferase (SPT) [6]. Surprisingly, these increases are paralleled by increased appearance of radiolabeled ceramide in circulating lipoproteins, indicating that part of the ceramide generated de novo in the liver, is secreted via VLDL/LDL
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pathway [6,7]. Indeed, isolated rat hepatocytes secrete ceramide as a component of very-low-density lipoproteins (VLDL) and low-density lipoproteins (LDL) [8]. Importantly, changes in the hepatic SPT activity, the rate-limiting step in de novo ceramide synthesis, affect the rate of ceramide secretion. Activation of SPT by the addition of palmitic acid but not other fatty acids leads to an elevation in VLDL and LDL ceramide. Vice versa, inhibition of de novo ceramide synthesis by Fumonisin B1 prevents the incorporation of ceramide in VLDL and LDL without affecting the overall lipoprotein synthesis and the secretion of ApoB [8]. These studies provide first evidence that hepatocytes secrete ceramide in a regulated fashion. More recent reports have proposed a second mechanism for the regulation of ceramide content of lipoproteins. It involves the secreted SMase (SSMase), that is product of acid SMase gene, and hydrolyses SM in the LDL particles to ceramide [9]. The enzyme is secreted by vascular endothelial cells and by activated macrophages in response to IL-1b and TNFa in vitro [10] and LPS in vivo [11]. Taken together, these studies show that ceramide is component of circulating lipoproteins and its level is regulated during host response to inflammation. At present, the physiological significance of these observations is unclear. It has been shown that increases in ceramide content affect the biophysical properties and structure of LDL. For example, LDL treated with SMase in vitro aggregates at a higher rate and tends to be retained in the vascular wall [9]. Indeed, aggregated LDL extracted from human aortic plaques has increased ceramide content as compared to monomeric LDL. In addition, the turnover of LDL SM to ceramide enhances the rate of LDL oxidation due to disordering effects in the lipid moiety and increased accessibility to reactive oxygen species [12]. Finally, it is conceivable that the uptake of lipoproteins with elevated ceramide content may represent an alternative pathway for increasing cellular ceramide concentrations in cells with active lipoprotein turnover, such as vascular endothelium and macrophages. Experimental evaluation of the role of ceramide in circulation however is difficult, since information regarding ceramide in human lipoproteins is lacking. Direct extrapolation of the existing animal data to humans would be inaccurate because of the distinctive distribution and metabolism of circulating lipoproteins in humans. The goals of the present study are: (i) to examine the presence and distribution of ceramide in lipoproteins from humans; (ii) to determine whether the acute phase response to inflammation increases plasma levels of ceramide in humans; and (iii) using animal models, to evaluate the roles of de novo synthesis of ceramide and secreted SMase for the elevation of serum ceramide.
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Materials and methods Materials N-hexanoyl-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-sphingosine phosphorylcholine (C6 -NBDSM)was purchased from Matreya (State College, PA), C2 –C20 ceramide (N-acetyl Sphinganine) was synthesized as described previously [5]. 3 H-labeled L -serine was from American Radiolabeled Chemicals (St. Louis, MO). All other reagents were from Sigma Chemical (St. Louis, MO). Study design Eleven healthy male volunteers (21–44 years of age) were enrolled in this study. The protocol was approved by the University of Kentucky Institutional Review Board and the General Clinical Research Center Committee and was identical to the one used in previous studies [13,14]. Written consent was obtained at enrollment. All subjects were healthy as confirmed by medical history, physical exam, blood chemistry, urinalysis, and ECG. They were non-smokers and were advised to ingest no alcohol, caffeine, non-steroidal anti-inflammatory drugs and other medications for at least 3 days (7 days for the alcohol use) prior to and throughout the study. Each subject underwent two separate studies: one after saline administration and another following the administration of Escherichia coli lipopolysaccharide (LPS, US Standard Reference Endotoxin, CC-RE Lot 2). The order of treatments was randomized and in a balanced crossover design, separated by a 10- to -20 days washout period. After overnight fasting, each participant received LPS (20 endotoxin Units/kg. b.w.) or saline via intravenous injection at 8:00 a.m. Blood was collected at 0, 1.5, 3.0, 6.0, and 24.0 h after the treatments. Cell-free plasma was isolated and frozen at )70 °C for future analyses. For three of the subjects in the study, fresh plasma was used to isolate lipoproteins as described below. To confirm the inflammatory response, clinical signs such as heart rate, body temperature, and blood pressure were monitored throughout the study. The levels of plasma TNF-a, IL-6, and IL-10 were determined by commercially available kits (Quantikine; R&D Systems, Minneapolis, MN). Serum C reactive protein levels were determined by commercial kit from Behring Diagnostic (Somerville, NJ). Isolation of lipoprotein fractions For analyses of the distribution of sphingolipids in the plasma, 250 ml of human plasma (Kentucky Blood Bank) was used. Lipoproteins were separated by sequential ultracentrifugaion at 45,000 rpm (Beckman Ti70 rotor) for 18 h at densities 1.019, 1.063, and 1.25 to
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obtain VLDL, LDL, and high-density lipoproteins (HDL), respectively. Lipoproteins were also isolated from 2 ml of human plasma according to the same method, however the centrifugations were performed at 70,000 rpm (Beckman rotor TLA100.2) for 3 h using a Beckman Optra tabletop ultracentrifuge. For quality control, the isolated lipoprotein fractions were analyzed by polyacrylamide gel electrophoresis.
separation, the aqueous phase was removed and the organic phase was washed again with 2 ml of basic water to remove any traces of radiolabeled serine. The radioactivity in the organic phase was measured by scintillation counter and SPT specific activity was calculated based on the specific labeling of the substrate. Samples without microsomal protein were run through the procedure and used to correct for background.
Animals and treatments
Assay of S-SMase activity in mouse plasma
LDL receptor knockout mice, LDLRKO ()/)), were purchased from Jackson Laboratory (Bar Harbor, Maine). A colony of acid sphingomyelinase knockout (ASMKO) mice was maintained in the AAALAC-approved animal facility of University of Kentucky Medical Center by breeding ASMKO ()/+) mice. After weaning, the mice were genotyped [15] and placed on a standard NIH-31 diet, 12-h light/dark cycle in microisolation. Littermate ASMKO (+/+) mice were used as controls for ASMKO ()/)) mice throughout the experiments. Male C57/BL6, LDLRKO ()/)), ASMKO (+/+), and ASMKO ()/)) mice were injected i.p. with LPS (5.8 mg/kg. b.w., E. coli 011:b4, Difco, BD Bioscience, Franklin Lakes, NJ) or with 150 mM NaCl. After different times, the mice were deeply anesthetized, blood was collected by heart puncture, and the serum was obtained by allowing the blood to coagulate for 2 h at 4 °C, followed by centrifugation at 14,000 rpm in a refrigerating Eppendorf microfuge. Liver was excised and used immediately for isolation of microsomes.
S-SMase activity was assayed as described by Wong et al. [11]; however, LDL labeled with N-hexanoyl-(N(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-sphingosine- phosphorylcholine (C6 -NBD-SM) was used as a substrate. The substrate was prepared as described earlier [17]. The hydrolysis of NBD-SM and the formation of NBD-ceramide were quantified by HPLC after calibration with external standards [5].
Assay of serine–palmitoyl transferase (SPT) activity [16] For analyses of SPT activity, equal amounts of liver tissue from 5 mice were combined (one lob per liver was used) and 10% homogenates were prepared in 100 mM Tris–HCl, pH 7.4, using motor-driven homogenizer. The 500 g supernatant of the homogenate was centrifuged at 20,000g for 10 min and the supernatant was centrifuged for 1 h at 105,000g. The pellet was resuspended in 100 mM Tris buffer (pH 7.4), aliquoted, and frozen for future use. The SPT activity was analyzed in 100 mM Hepes (pH 8.3) 5 mM dithiothreitol, 2.5 mM EDTA (pH 7), 50 lM pyridoxal phosphate, 200 lM palmitoyl-CoA, 1 mM 3 H-labeled L -serine (specific activity of 10–30 mCi/ mmol), and 100 lg microsomal protein. The reaction was initiated by the addition of palmitoyl–CoA to avoid depletion of the substrate by the fatty acid CoA hydrolyses. The reaction was incubated at 37 °C with shaking for 10 min. The radiolabeled enzymatic product, 3-ketosphinganine, was separated from the radiolabeled substrate by phase partitioning in 1 ml CHCl3 and 2 ml of 0.5 N NH3 OH. Sphinganine (25 ll from 1 mg/ml stock solution in ethanol) was used as a carrier. After phase
Measurements of sphingolipid mass The lipids were extracted by the method of Blight and Dyer [18], modified as described previously [19], and analyzed by thin-layer chromatography on silica gel 60 plates (10 20 cm) using chloroform: methanol: triethylamine: 2-propanol: 0.25% potassium chloride (30:9:18: 25:6, by volume) as the developing solvent. The regions migrating with a standard ceramide (bovine brain, Matreya, Pleasant Gap, PA) were scraped from the plate. To quantify the mass of ceramide, the lipids were eluted from the silica with 1 ml chloroform: methanol (1:1, by vol.) followed by 1 ml methanol. The combined eluates were dried in vacuo, then 0.5–1.0 nmol of internal standard, N-acetyl-C20 -sphinganine, was added to the unknown ceramide sample, and the ceramide mass was quantified by HPLC of the long-chain bases released after an acid hydrolysis in 0.5 M HCl in methanol at 65 °C for 15 h. Free long-chain bases were analyzed as described by Merrill et al. [20]. Statistical analysis Repeated measures ANOVA with DunnetÕs post hoc analysis were used to compare the changes in vital signs and cytokine in LPS vs. control treated subject. The other statistical analyses were performed using unpaired StudentÕs t test.
Results Plasma ceramide is carried mainly via VLDL and LDL Despite the increasing evidence for the role of ceramide on the aggregation and oxidation state of serum
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lipoproteins, information regarding the ceramide content of lipoproteins in normal and patho-physiological state is lacking. As far as one can readily ascertain, the only published study to quantify the mass of ceramide in serum is done in rats that differ greatly from humans in respect to their lipoprotein profiles [8]. Therefore, we determined the sphingolipid composition of VLDL, LDL, HDL, and lipoprotein-deficient serum (LPDS) isolated from plasma of healthy humans. The quality of the isolated lipoproteins was controlled by gel electrophoresis and the lack of albumin contamination was confirmed (data not shown). About 80% of the total plasma ceramide was associated with VLDL and LDL, and only 5 % was in HDL (Fig. 1). VLDL and LDL fractions also had the highest concentration of ceramide per milligram of protein that ranged from 2 to 4 nmol/ mg. protein (Table 1). The LPDS fraction, which consists mainly of albumin, carried only about 15% of
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serum ceramide. Dihydroceramide and sphingosine had similar distribution, however, LPDS accounted for 25% of their total plasma levels. In a sharp contrast, LPDS carried almost a half of serum dihydrosphingosine (sphinganine). The cause for the differential distribution of sphinganine is not known; however, it may reflect the fact that sphinganine is synthesized in the endoplasmic reticulum [21] and therefore may co-localize with the site for albumin synthesis. In turn, the synthesis of more complex sphingolipids, such as ceramide and sphingomyelin, occurs at the Golgi apparatus [22], where the lipid shell of VLDL is assembled. Induction of acute inflammation in humans leads to increases in plasma ceramide mass LPS-induced APR in rodents is associated with increases in the hepatic ceramide level [1,4–7]. Studies in
Fig. 1. Distribution of sphingolipids among plasma lipoproteins. Individual lipoprotein fractions were isolated from human plasma obtained from healthy blood donors (Kentucky Blood Bank). The mass of sphingolipids was determined by TLC/HPLC method. The level of each sphingolipid is presented as percent from its level in total plasma after corrections for the yield of the lipoproteins. All assays were performed in triplicate with standard deviation less then 10%. The data are representative for 2 donors tested.
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Table 1 Sphingolipid content of human lipoproteins (nmol/mg protein)
VDVL LDL HDL LPDS
Ceramide
Dihydro-ceramide
Sphingosine
Dihydro-sphingosine
3.783 0.310 2.927 0.125 0.286 0.123 0.013 0.003
0.452 0.002 0.523 0.006 0.048 0.009 0.006 0.001
0.153 0.080 0.210 0.040 0.073 0.040 0.004 0.001
0.074 0.036 0.084 0.005 0.026 0.001 0.004 0.001
Lipoproteins were isolated by density centrifugation from human plasma. The mass of the indicated spingolipids was determined by TLC/HPLC and normalized for the amount of protein in each lipoprotein fraction. Data are average SD (n ¼ 3) and are representative for diferent individuals.
hamster model have shown an increased labeling of lipoprotein-associated ceramide in response to LPS, suggesting that part of the excess ceramide generated during APR in the liver is secreted into the circulation [6]. These data imply that ceramide mass in lipoproteins may increase during APR. To test this, we used a human model of inflammation. An i.v. administration of E. coli lipopolysaccharide (LPS, US Standard Reference Endotoxin, CC-RE-Lot2) was used as a safe reproducible model of sepsis. LPS (20 EU/kg. b.w.) was administered to healthy male volunteers and serum samples were collected. In a separate study, each subject received saline injection instead of LPS that served as control. All subjects began to experience fever, chills, headache, malaise, and mild nausea beginning approximately 1.5 h after the LPS administration. To confirm the inflammatory response heart rate, body temperature, blood pressure, the levels of pro-inflammatory cytokines TNF-a and IL-6, and the levels of C-reactive protein were monitored throughout the study. The changes in these indices documented the induction of acute inflammatory response in all volunteers. The detailed clinical data have been published previously [13,14,23]. The mass of ceramide in serum was assayed in eight subjects at 0, 1.5, 3, 6, and 24 h after saline or LPS administration. The measurements were done in triplicates for each time point. Initial analyses on the efficiency of the extraction of ceramide from plasma showed that the procedure was linear when up to 50 ll was used (data not shown). The values obtained for different times after LPS were normalized for the values at the respective time after the saline injection. Data were presented as fold changes over the value at time ‘‘0.’’ Such representation allowed accounting for differences in the lipoprotein profiles of the individual subjects. Statistically significant increases were found in six out of the eight individuals tested for total plasma ceramide. One subject showed increases in ceramide level after saline injection alone and was excluded from the analyses. Among the patients with significant increases in the plasma ceramide level, two different patterns were found. One group consisting of three patients showed progressive elevation in ceramide beginning at 3 h after LPS administration and continuing for up to 24 h (Fig. 2, upper panel). The magnitude of these changes ranged from 2.5- to 10-fold.
A second group of patients experienced transient increases. These changes were with smaller magnitude ranging between 30% and 4-fold, peaked at 1.5 h and return to normal levels, or in one case decreased, thereafter (Fig. 2, lower panel). The mechanism to explain these differences in the human population is unclear. One possible explanation is that they originate from differences in the systemic response to LPS administration. However, no correlations were found between the pattern of ceramide increases and the changes in the clinical indices. Alternatively, the appearance of two patterns of response may reflect differences in lipoprotein profile of the patients and/or in the rate of VLDL/LDL metabolism. LPS-induced increases in ceramide are associated with VLDL and LDL particles Two pathways may increase plasma ceramide levels during APR: (i) changes in the secretion of ceramide by the liver due to increases in its de novo synthesis; and (ii) activation of secreted SMase that catalyzes the conversion of SM in the LDL to ceramide. If de novo synthesis of ceramide is activated, the levels of ceramide in both, VLDL and LDL, should increase. Activation of SSMase however is likely to affect only ceramide levels in LDL because the enzyme does not act on SM in VLDL particles. To test this, the lipoprotein fraction(s) with elevated ceramide concentration was identified. Lipoproteins were isolated from fresh plasma obtained from 3 of the 11 subjects in the study and ceramide in VLDL, LDL, and HDL was quantified. The levels of ceramide in HDL were not affected by LPS administration. Up to twofold increases in ceramide levels were found in both, VLDL and LDL, fractions (Fig. 3) in all three participants. Surprisingly, the changes in VLDL appeared earlier than the changes in LDL: maximum increases in VLDL were found at 6 h, while the changes in LDL occurred at 24 h after LPS administration. Such pattern implies that early activation of de novo synthesis of ceramide in the liver may contribute to the increases in VLDL ceramide, while the delayed elevation in LDL ceramide may be a result from a normal metabolism of VLDL to LDL and/or from activation of secreted SMase. Furthermore, the differential increases of
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Fig. 2. LPS administration in humans increases the concentration of ceramide in the plasma. The data for each individual (total of 7 subjects shown) are represented with specific hatch pattern or shade. The volunteers were injected i.v. with saline or LPS (20 U per kg. b.w.) in two separate randomized studies separated by 10–20 days of a washout period. Blood was collected at the indicated times after LPS and saline injection. The levels of ceramide in the plasma were measured by TLC/HLPC method and the values at different times after LPS were normalized for the values at the respective time after the saline injection. Saline injection alone did not cause significant changes but there were individual-to-individual differences in the basal levels of ceramide. To account for this, the data are presented as a fold change over the value at time ‘‘0.’’ Based on the different patterns of response to LPS, the subjects were grouped into two groups presented in panels A and B. The ceramide assays were performed in duplicate and the standard deviation was less than 10%. *, p < 0:05; **, p < 0:005.
ceramide in VLDL and LDL fractions may contribute to the appearance of two different patterns of LPS response in regard to the total plasma ceramide levels. LPS induces elevation in serum ceramide in mouse model
Fig. 3. LPS-induces increases in VLDL and LDL ceramide content. Fresh plasma from 3 additional volunteers was used to isolate lipoprotein fractions. Treatments were conducted as described in the legend for Fig. 2. The levels of ceramide were determined by TLC/HPLC method. The data were normalized per the amount of protein present in the lipoproteins, represent average SD, for three ceramide assays, and are representative for the three subjects tested. *, p < 0:05.
The mechanism(s) involved in the elevation of serum ceramide by LPS were tested in a mouse model further. Due to the differences in lipoprotein profiles of humans and mice, two mouse strains were used: C57/Bl6 and LDL receptor (LDLR) knockout mice [24]. The former have very low levels of LDL in the serum. In contrast, the latter lack LDL receptor, they have substantially increased LDL level in the circulation, and resemble lipoprotein profile in humans. LPS was administered i.p. and serum was collected 20 h after the injection. In the C57/Bl6 mice, there was a moderate increase in ceramide levels in total serum that reached 35% over the value for
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Table 2 Elevation in serum ceramide levels in different mouse models
C57/BL6 LDLRKO
Cer DHCer Cer DHCer
Control
LPS
3.49 1.15 ND 7.92 1.51 1.67 0.60
4.74 0.22 ND 12.41 2.16 1.65 0.10
Mice were injected i.p. with LPS and sacrificed after 20 h. Data are expressed as nmol/ml serum and are average SD (n ¼ 3 animals per group). Animal-to-animal deviations are shown.
saline-injected mice (Table 2). In the LDLR knockout mice, the basal and LPS-induced levels were significantly higher than in C57/Bl6, reflecting the higher number of LDL particles. Moreover, the LPS-induced increases were somewhat higher, reaching 60% of the control value.
SPT at 6 h after the injection (Fig. 4). Later time points (20 h post-LPS) showed no differences in SPT activity (not shown). Simultaneous to the activation of SPT, LPS administration also led to increase in S-SMase activity of (Fig. 5, panel A), thus suggesting that both enzymes may contribute for elevating serum ceramide levels. To distinguish between these two possibilities, ASMKO mice were used. These mice lack the gene for ASMase, which encodes the lysosomal sphingomyelinase (L-SMase), as well as the S-SMase. At the same time LPS-induced cytokine secretion including that of TNFa and IL-6 is normal [25]. If LPS administration does not elevate serum ceramide in ASMKO ()/)) mice, then S-SMase is
LPS administration to mice activates both, SPT activity and S-SMase activity Data from earlier studies suggest two possible mechanisms for the elevation of VLDL and LDL ceramide during APR: (i) Stimulation of SPT and de novo synthesis in liver as proposed in hamsters [6,7] and (ii) An increased conversion of LDL SM to ceramide suggested by the increased activity of SSMase in serum of LPS-injected mice [11]. To test the role of each pathway, the activity of SPT in isolated liver microsomes and that of S-SMase in serum were measured. LPS administration led to significant increase in the specific activity of
Fig. 4. Activation of serine–palmitoyl transferase activity in liver microsomes of mice treated with saline and LPS. Mice (n ¼ 5) were injected with LPS (5.8 mg/kg b.w.) or with saline and sacrificed 6 h latter. Livers were excised, equal parts were pooled together, and microsomes were isolated at 105,000g. serine–palmitoyl transferase activity was analyzed in vitro using Palmitoyl CoA and 3 H-labeled L -serine as substrates. The enzymatic product, 3-keto-sphinganine, was separated from the radiolabeled substrate by phase partitioning and measured by scintillation counter. The specific activity was expressed as pmol/mg. Pr/h. Data are average SD (n ¼ 3) and are representative of 2 independent analyses. *, p < 0:05.
Fig. 5. Administration of LPS to ASMKO ()/)) mice causes increases in serum ceramide levels in the absence of S-SMase activity. Male ASMKO (+/+) and ASMKO ()/)) were injected with LPS or saline as described in the legend for Fig. 4. S-SMase activity and ceramide levels in serum were quantified as described in Materials and methods. The data are average SD and animal-to-animal deviations are shown (n ¼ 3 animals per group). *, p < 0:05.
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likely the only enzyme involved. In turn, if the increases in serum ceramide levels are similar in ASMKO ()/)) and ASMKO (+/+) mice, then de novo synthesis of ceramide in the liver is also involved. This result however does not eliminate the role of SSMase completely because ASMKO mice are in C57Bl6 background, and the substrate for S-SMase, LDL, is rapidly cleared from the circulation, thus preventing the detection of differences in ceramide in LDL. Having in mind these limitations, ASMKO ()/)) and litter-matched ASMKO (+/+) mice were injected with LPS or saline. As expected, LPS administration induced activation of S-SMase and elevation of serum ceramide in ASMKO (+/+) mice. In contrast, ASMKO ()/)) had no detectable S-SMase activity in neither saline- nor LPS-injected mice, thus confirming that ASMase is the only secreted, LPS-activated, SMase activity (Fig. 5, panel A). In spite of the lack of SSMase, LPS administration led to elevation in serum ceramide, although the basal levels were somewhat lower than in ASMKO (+/+) mice (Fig. 5, panel B). This indicates that activation of de novo synthesis of ceramide in the liver contributes to the increases in serum ceramide during inflammation.
Discussion This manuscript presents evidence that APR in humans and mice is associated with increases in the mass of ceramide in plasma. Using a human model and mass analyses, our experiments confirm and extend the results obtained earlier in a hamster model (15, 16). The major carriers of ceramide in the plasma are VLDL and LDL; respectively, the LPS administration increased exclusively VLDL and LDL ceramide content. These increases are likely to affect LDL functions in a couple of different ways. One possibility is that elevation of LDL ceramide content may affect the biophysical properties of the particles. Indeed, treatment of gigantic unilamellar liposomes [26] and LDL [9] with sphingomyelinase generates excess ceramide that self-aggregates in ceramide-enriched domains, leading to the aggregation and fusion of the particles. In turn, aggregation of LDL particles may contribute to the development of atherosclerotic plaques. Second, the up-take of ceramide-enriched LDL by the cells of the vasculature may lead to accumulation of ceramide inside the cells. Such accumulation may have an effect on cell functions similar to that mediated by agonist-induced activation of intracellular sphingomyelinases. For example, the up-take of LDL with elevated ceramide content by endothelial cells causes accumulation of ceramide and increases the incidence of apoptosis [17]. Interestingly, increased rate of apoptosis in microvascular endothelium underlies the loss of blood pressure and vascular
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tone during severe endotoxic shock. In addition, the exogenously delivered ceramide may be converted to sphingosine [17] and sphingosine-phosphate, potent proinflammatory second messenger molecules that are implicated in the regulation of adhesion molecule expression [27]. Finally, a third possibility is that by binding to the different LDL-binding receptors, ceramide-rich LDL particles deliver excess ceramide to the plasma membranes, particularly to the lipid rafts. Notably, the formation of ceramide-rich lipid rafts has been suggested to play a role in host response to infection [28]. This manuscript also provides novel evidence for the role of liver SPT during inflammation. Activation of SPT has been correlated with increased production of ceramide inside the cells and induction of apoptosis or cell growth arrest [29]. In lower eukaryotes, for example, SPT is involved in the regulation of heat stress response and yeast strains with mutation in LCB1, one of the two genes encoding SPT, lack the ability to adapt to elevation in temperature [30]. In mammalian cells, activation of SPT plays a role in mediating the responses to different inducers or regulators of cellular apoptosis and growth arrest [31,32]. Data began to accumulate that in the liver, SPT may play an additional role, namely to regulate the rate of ceramide secretion in the circulation. The evidence for such a role can be summarized as follows: (i) In Syrian hamster model, activation of SPT by LPS has been described and correlated to the increases in the secretion of ceramide in plasma lipoproteins. (ii) The data from ASMKO ()/)) mice presented in this manuscript provide additional evidence that increases in serum ceramide levels are mediated via activation of SPT, rather than solely due to activation of SSMase; (iii) SPT is the rate-limiting step in de novo ceramide synthesis and secretion in liver, and activation of SPT in isolated primary hepatocytes causes increases in ceramide level in VLDL [8], while inhibition of de novo synthesis of ceramide by Fumonisin B1 prevents its secretion by hepatocytes. The mechanism for activation of SPT during APR is not completely understood. LPS administration in hamster significantly increases the level of SPT mRNA in the liver in correlation with the increases in its activity [6]. Similar changes are reported in response to cytokines, such as IL-1b, as well as UV irradiation. These observations suggest that transcriptional regulation may be a common mechanism for activation of SPT in proinflammatory conditions; however, the transcription factors involved are not known. The physiological function of regulated ceramide secretion by the liver is unclear. It is possible that the ability to secrete ceramide is a unique feature of that organ related to its role in lipoprotein synthesis and secretion. Stimulation of ceramide release during inflammation may be part of the mechanism by which liver contributes to the dysfunction of vascular endo-
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thelium during APR to infections. Thus, it is possible that in addition to its role of intracellular lipid mediator, ceramide acts as a systemic mediator of inflammation. Alternatively, the regulated secretion of ceramide could be a mechanism for ceramide removal and cell protection during systemic stress.
Acknowledgments This work was supported partly by research award from the American Federation for Aging Research (to M.N.-K) and a Grant-in-Aid from the American Heart Association (Ohio Valley affiliate (to M.N.-K), as well as by the Department of Veterans Affairs Merit Award Program (to S.I.S.) and by M01RR02602 NIH Grant for the University of Kentucky General Clinical Research Center (to S.I.S.). We greatly appreciate the comments of the Cardiovascular Research Group.
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Elevation of ceramide in serum lipoproteins during acute phase response in humans and mice: role of serine–palmitoyl transferase Sandy Lightle,a Raina Tosheva,b Amy Lee,a Jennie Queen-Baker,a,b,c Boris Boyanovsky,a Steve Shedlofsky,b,c and Mariana Nikolova-Karakashiana,* a Department of Physiology, University of Kentucky Lexington, KY 40536, USA Department of Medicine, VA Medical Center, University of Kentucky Lexington, KY 40536, USA General Clinical Research Center, College of Medicine, University of Kentucky Lexington, KY 40536, USA b
c
Received 27 January 2003, and in revised form 15 August 2003
Abstract Recent studies have indicated that ceramide generated in the liver is secreted into the bloodstream as component of very-lowdensity lipoproteins (VLDL) and low-density lipoproteins (LDL). This manuscript investigates the effect of host acute phase response to inflammation on lipoprotein ceramide levels. In humans, two different patterns of responses were found. One group of volunteers experienced transient increases in serum ceramide at 1.5 h after LPS administration. Second group showed prolonged increases that reached up to 10-fold above the basal level and continued for up to 24 h. Increases in ceramide were found only in VLDL and LDL particles. LPS administration induced similar increases in mice. These increases were accompanied by activation of secreted sphingomyelinase in serum and serine–palmitoyl transferase in liver. ASMase knockout mice retained LPS-induced increases in serum ceramide, thus suggesting that the elevation of VLDL and LDL ceramide content is attributed at least in part to activation of de novo synthesis of ceramide in the liver. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Atherosclerosis; LPS; Liver; Sphingomyelinase; Sphingosine
Ceramide, a sphingolipid second messenger molecule, has been extensively studied for its role in cellular response to stress and inflammation. Key inducers and mediators of host acute phase response (APR),1 such as bacterial lipopolysaccharide (LPS), Interleukin 1b (IL1b), and Tumor necrosis factor a (TNFa), induce early generation of ceramide in the liver [1–3]. In isolated hepatocyte cultures, the accumulation of ceramide occurs within 1 h of exposure to IL-1b and is caused by activation of a neutral sphingomyelinase (SMase), an
*
Corresponding author. Fax: 1-606-323-1070. E-mail address: [email protected] (M. Nikolova-Karakashian). 1 Abbreviations used: VLDL, very-low-density lipoproteins; LDL, low-density lipoproteins; LPS, lipopolysaccharide; IL-1b, interleukin 1b; TNFa, tumor necrosis factor a; SMase, sphingomyelinase; SPT, serine–palmitoyl transferase; SSMase, secreted SMase; LPDS, lipoprotein-deficient serum; LDLR, LDL receptor. 0003-9861/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2003.08.031
enzyme that metabolizes SM in cellular membranes to phosphorylcholine and ceramide. The latter appears to mediate the cytokine-induced expression of serum acute phase proteins, including a1 -acid glycoprotein, C-reactive protein, and serum amyloid A [1,4]. The agonistinduced activation of sphingomyelinase is often coupled to activation of ceramidase and sphingosine kinase that convert ceramide to other bioactive sphingolipids, sphingosine and sphingosine-phosphate, thus restoring the normal cellular levels of ceramide [5]. Studies in a hamster model of inflammation suggest that in addition to activation of sphingomyelinase, liver APR leads to increases in de novo synthesis of ceramide via activation of serine–palmitoyl transferase (SPT) [6]. Surprisingly, these increases are paralleled by increased appearance of radiolabeled ceramide in circulating lipoproteins, indicating that part of the ceramide generated de novo in the liver, is secreted via VLDL/LDL
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pathway [6,7]. Indeed, isolated rat hepatocytes secrete ceramide as a component of very-low-density lipoproteins (VLDL) and low-density lipoproteins (LDL) [8]. Importantly, changes in the hepatic SPT activity, the rate-limiting step in de novo ceramide synthesis, affect the rate of ceramide secretion. Activation of SPT by the addition of palmitic acid but not other fatty acids leads to an elevation in VLDL and LDL ceramide. Vice versa, inhibition of de novo ceramide synthesis by Fumonisin B1 prevents the incorporation of ceramide in VLDL and LDL without affecting the overall lipoprotein synthesis and the secretion of ApoB [8]. These studies provide first evidence that hepatocytes secrete ceramide in a regulated fashion. More recent reports have proposed a second mechanism for the regulation of ceramide content of lipoproteins. It involves the secreted SMase (SSMase), that is product of acid SMase gene, and hydrolyses SM in the LDL particles to ceramide [9]. The enzyme is secreted by vascular endothelial cells and by activated macrophages in response to IL-1b and TNFa in vitro [10] and LPS in vivo [11]. Taken together, these studies show that ceramide is component of circulating lipoproteins and its level is regulated during host response to inflammation. At present, the physiological significance of these observations is unclear. It has been shown that increases in ceramide content affect the biophysical properties and structure of LDL. For example, LDL treated with SMase in vitro aggregates at a higher rate and tends to be retained in the vascular wall [9]. Indeed, aggregated LDL extracted from human aortic plaques has increased ceramide content as compared to monomeric LDL. In addition, the turnover of LDL SM to ceramide enhances the rate of LDL oxidation due to disordering effects in the lipid moiety and increased accessibility to reactive oxygen species [12]. Finally, it is conceivable that the uptake of lipoproteins with elevated ceramide content may represent an alternative pathway for increasing cellular ceramide concentrations in cells with active lipoprotein turnover, such as vascular endothelium and macrophages. Experimental evaluation of the role of ceramide in circulation however is difficult, since information regarding ceramide in human lipoproteins is lacking. Direct extrapolation of the existing animal data to humans would be inaccurate because of the distinctive distribution and metabolism of circulating lipoproteins in humans. The goals of the present study are: (i) to examine the presence and distribution of ceramide in lipoproteins from humans; (ii) to determine whether the acute phase response to inflammation increases plasma levels of ceramide in humans; and (iii) using animal models, to evaluate the roles of de novo synthesis of ceramide and secreted SMase for the elevation of serum ceramide.
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Materials and methods Materials N-hexanoyl-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino)-sphingosine phosphorylcholine (C6 -NBDSM)was purchased from Matreya (State College, PA), C2 –C20 ceramide (N-acetyl Sphinganine) was synthesized as described previously [5]. 3 H-labeled L -serine was from American Radiolabeled Chemicals (St. Louis, MO). All other reagents were from Sigma Chemical (St. Louis, MO). Study design Eleven healthy male volunteers (21–44 years of age) were enrolled in this study. The protocol was approved by the University of Kentucky Institutional Review Board and the General Clinical Research Center Committee and was identical to the one used in previous studies [13,14]. Written consent was obtained at enrollment. All subjects were healthy as confirmed by medical history, physical exam, blood chemistry, urinalysis, and ECG. They were non-smokers and were advised to ingest no alcohol, caffeine, non-steroidal anti-inflammatory drugs and other medications for at least 3 days (7 days for the alcohol use) prior to and throughout the study. Each subject underwent two separate studies: one after saline administration and another following the administration of Escherichia coli lipopolysaccharide (LPS, US Standard Reference Endotoxin, CC-RE Lot 2). The order of treatments was randomized and in a balanced crossover design, separated by a 10- to -20 days washout period. After overnight fasting, each participant received LPS (20 endotoxin Units/kg. b.w.) or saline via intravenous injection at 8:00 a.m. Blood was collected at 0, 1.5, 3.0, 6.0, and 24.0 h after the treatments. Cell-free plasma was isolated and frozen at )70 °C for future analyses. For three of the subjects in the study, fresh plasma was used to isolate lipoproteins as described below. To confirm the inflammatory response, clinical signs such as heart rate, body temperature, and blood pressure were monitored throughout the study. The levels of plasma TNF-a, IL-6, and IL-10 were determined by commercially available kits (Quantikine; R&D Systems, Minneapolis, MN). Serum C reactive protein levels were determined by commercial kit from Behring Diagnostic (Somerville, NJ). Isolation of lipoprotein fractions For analyses of the distribution of sphingolipids in the plasma, 250 ml of human plasma (Kentucky Blood Bank) was used. Lipoproteins were separated by sequential ultracentrifugaion at 45,000 rpm (Beckman Ti70 rotor) for 18 h at densities 1.019, 1.063, and 1.25 to
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obtain VLDL, LDL, and high-density lipoproteins (HDL), respectively. Lipoproteins were also isolated from 2 ml of human plasma according to the same method, however the centrifugations were performed at 70,000 rpm (Beckman rotor TLA100.2) for 3 h using a Beckman Optra tabletop ultracentrifuge. For quality control, the isolated lipoprotein fractions were analyzed by polyacrylamide gel electrophoresis.
separation, the aqueous phase was removed and the organic phase was washed again with 2 ml of basic water to remove any traces of radiolabeled serine. The radioactivity in the organic phase was measured by scintillation counter and SPT specific activity was calculated based on the specific labeling of the substrate. Samples without microsomal protein were run through the procedure and used to correct for background.
Animals and treatments
Assay of S-SMase activity in mouse plasma
LDL receptor knockout mice, LDLRKO ()/)), were purchased from Jackson Laboratory (Bar Harbor, Maine). A colony of acid sphingomyelinase knockout (ASMKO) mice was maintained in the AAALAC-approved animal facility of University of Kentucky Medical Center by breeding ASMKO ()/+) mice. After weaning, the mice were genotyped [15] and placed on a standard NIH-31 diet, 12-h light/dark cycle in microisolation. Littermate ASMKO (+/+) mice were used as controls for ASMKO ()/)) mice throughout the experiments. Male C57/BL6, LDLRKO ()/)), ASMKO (+/+), and ASMKO ()/)) mice were injected i.p. with LPS (5.8 mg/kg. b.w., E. coli 011:b4, Difco, BD Bioscience, Franklin Lakes, NJ) or with 150 mM NaCl. After different times, the mice were deeply anesthetized, blood was collected by heart puncture, and the serum was obtained by allowing the blood to coagulate for 2 h at 4 °C, followed by centrifugation at 14,000 rpm in a refrigerating Eppendorf microfuge. Liver was excised and used immediately for isolation of microsomes.
S-SMase activity was assayed as described by Wong et al. [11]; however, LDL labeled with N-hexanoyl-(N(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-sphingosine- phosphorylcholine (C6 -NBD-SM) was used as a substrate. The substrate was prepared as described earlier [17]. The hydrolysis of NBD-SM and the formation of NBD-ceramide were quantified by HPLC after calibration with external standards [5].
Assay of serine–palmitoyl transferase (SPT) activity [16] For analyses of SPT activity, equal amounts of liver tissue from 5 mice were combined (one lob per liver was used) and 10% homogenates were prepared in 100 mM Tris–HCl, pH 7.4, using motor-driven homogenizer. The 500 g supernatant of the homogenate was centrifuged at 20,000g for 10 min and the supernatant was centrifuged for 1 h at 105,000g. The pellet was resuspended in 100 mM Tris buffer (pH 7.4), aliquoted, and frozen for future use. The SPT activity was analyzed in 100 mM Hepes (pH 8.3) 5 mM dithiothreitol, 2.5 mM EDTA (pH 7), 50 lM pyridoxal phosphate, 200 lM palmitoyl-CoA, 1 mM 3 H-labeled L -serine (specific activity of 10–30 mCi/ mmol), and 100 lg microsomal protein. The reaction was initiated by the addition of palmitoyl–CoA to avoid depletion of the substrate by the fatty acid CoA hydrolyses. The reaction was incubated at 37 °C with shaking for 10 min. The radiolabeled enzymatic product, 3-ketosphinganine, was separated from the radiolabeled substrate by phase partitioning in 1 ml CHCl3 and 2 ml of 0.5 N NH3 OH. Sphinganine (25 ll from 1 mg/ml stock solution in ethanol) was used as a carrier. After phase
Measurements of sphingolipid mass The lipids were extracted by the method of Blight and Dyer [18], modified as described previously [19], and analyzed by thin-layer chromatography on silica gel 60 plates (10 20 cm) using chloroform: methanol: triethylamine: 2-propanol: 0.25% potassium chloride (30:9:18: 25:6, by volume) as the developing solvent. The regions migrating with a standard ceramide (bovine brain, Matreya, Pleasant Gap, PA) were scraped from the plate. To quantify the mass of ceramide, the lipids were eluted from the silica with 1 ml chloroform: methanol (1:1, by vol.) followed by 1 ml methanol. The combined eluates were dried in vacuo, then 0.5–1.0 nmol of internal standard, N-acetyl-C20 -sphinganine, was added to the unknown ceramide sample, and the ceramide mass was quantified by HPLC of the long-chain bases released after an acid hydrolysis in 0.5 M HCl in methanol at 65 °C for 15 h. Free long-chain bases were analyzed as described by Merrill et al. [20]. Statistical analysis Repeated measures ANOVA with DunnetÕs post hoc analysis were used to compare the changes in vital signs and cytokine in LPS vs. control treated subject. The other statistical analyses were performed using unpaired StudentÕs t test.
Results Plasma ceramide is carried mainly via VLDL and LDL Despite the increasing evidence for the role of ceramide on the aggregation and oxidation state of serum
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lipoproteins, information regarding the ceramide content of lipoproteins in normal and patho-physiological state is lacking. As far as one can readily ascertain, the only published study to quantify the mass of ceramide in serum is done in rats that differ greatly from humans in respect to their lipoprotein profiles [8]. Therefore, we determined the sphingolipid composition of VLDL, LDL, HDL, and lipoprotein-deficient serum (LPDS) isolated from plasma of healthy humans. The quality of the isolated lipoproteins was controlled by gel electrophoresis and the lack of albumin contamination was confirmed (data not shown). About 80% of the total plasma ceramide was associated with VLDL and LDL, and only 5 % was in HDL (Fig. 1). VLDL and LDL fractions also had the highest concentration of ceramide per milligram of protein that ranged from 2 to 4 nmol/ mg. protein (Table 1). The LPDS fraction, which consists mainly of albumin, carried only about 15% of
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serum ceramide. Dihydroceramide and sphingosine had similar distribution, however, LPDS accounted for 25% of their total plasma levels. In a sharp contrast, LPDS carried almost a half of serum dihydrosphingosine (sphinganine). The cause for the differential distribution of sphinganine is not known; however, it may reflect the fact that sphinganine is synthesized in the endoplasmic reticulum [21] and therefore may co-localize with the site for albumin synthesis. In turn, the synthesis of more complex sphingolipids, such as ceramide and sphingomyelin, occurs at the Golgi apparatus [22], where the lipid shell of VLDL is assembled. Induction of acute inflammation in humans leads to increases in plasma ceramide mass LPS-induced APR in rodents is associated with increases in the hepatic ceramide level [1,4–7]. Studies in
Fig. 1. Distribution of sphingolipids among plasma lipoproteins. Individual lipoprotein fractions were isolated from human plasma obtained from healthy blood donors (Kentucky Blood Bank). The mass of sphingolipids was determined by TLC/HPLC method. The level of each sphingolipid is presented as percent from its level in total plasma after corrections for the yield of the lipoproteins. All assays were performed in triplicate with standard deviation less then 10%. The data are representative for 2 donors tested.
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Table 1 Sphingolipid content of human lipoproteins (nmol/mg protein)
VDVL LDL HDL LPDS
Ceramide
Dihydro-ceramide
Sphingosine
Dihydro-sphingosine
3.783 0.310 2.927 0.125 0.286 0.123 0.013 0.003
0.452 0.002 0.523 0.006 0.048 0.009 0.006 0.001
0.153 0.080 0.210 0.040 0.073 0.040 0.004 0.001
0.074 0.036 0.084 0.005 0.026 0.001 0.004 0.001
Lipoproteins were isolated by density centrifugation from human plasma. The mass of the indicated spingolipids was determined by TLC/HPLC and normalized for the amount of protein in each lipoprotein fraction. Data are average SD (n ¼ 3) and are representative for diferent individuals.
hamster model have shown an increased labeling of lipoprotein-associated ceramide in response to LPS, suggesting that part of the excess ceramide generated during APR in the liver is secreted into the circulation [6]. These data imply that ceramide mass in lipoproteins may increase during APR. To test this, we used a human model of inflammation. An i.v. administration of E. coli lipopolysaccharide (LPS, US Standard Reference Endotoxin, CC-RE-Lot2) was used as a safe reproducible model of sepsis. LPS (20 EU/kg. b.w.) was administered to healthy male volunteers and serum samples were collected. In a separate study, each subject received saline injection instead of LPS that served as control. All subjects began to experience fever, chills, headache, malaise, and mild nausea beginning approximately 1.5 h after the LPS administration. To confirm the inflammatory response heart rate, body temperature, blood pressure, the levels of pro-inflammatory cytokines TNF-a and IL-6, and the levels of C-reactive protein were monitored throughout the study. The changes in these indices documented the induction of acute inflammatory response in all volunteers. The detailed clinical data have been published previously [13,14,23]. The mass of ceramide in serum was assayed in eight subjects at 0, 1.5, 3, 6, and 24 h after saline or LPS administration. The measurements were done in triplicates for each time point. Initial analyses on the efficiency of the extraction of ceramide from plasma showed that the procedure was linear when up to 50 ll was used (data not shown). The values obtained for different times after LPS were normalized for the values at the respective time after the saline injection. Data were presented as fold changes over the value at time ‘‘0.’’ Such representation allowed accounting for differences in the lipoprotein profiles of the individual subjects. Statistically significant increases were found in six out of the eight individuals tested for total plasma ceramide. One subject showed increases in ceramide level after saline injection alone and was excluded from the analyses. Among the patients with significant increases in the plasma ceramide level, two different patterns were found. One group consisting of three patients showed progressive elevation in ceramide beginning at 3 h after LPS administration and continuing for up to 24 h (Fig. 2, upper panel). The magnitude of these changes ranged from 2.5- to 10-fold.
A second group of patients experienced transient increases. These changes were with smaller magnitude ranging between 30% and 4-fold, peaked at 1.5 h and return to normal levels, or in one case decreased, thereafter (Fig. 2, lower panel). The mechanism to explain these differences in the human population is unclear. One possible explanation is that they originate from differences in the systemic response to LPS administration. However, no correlations were found between the pattern of ceramide increases and the changes in the clinical indices. Alternatively, the appearance of two patterns of response may reflect differences in lipoprotein profile of the patients and/or in the rate of VLDL/LDL metabolism. LPS-induced increases in ceramide are associated with VLDL and LDL particles Two pathways may increase plasma ceramide levels during APR: (i) changes in the secretion of ceramide by the liver due to increases in its de novo synthesis; and (ii) activation of secreted SMase that catalyzes the conversion of SM in the LDL to ceramide. If de novo synthesis of ceramide is activated, the levels of ceramide in both, VLDL and LDL, should increase. Activation of SSMase however is likely to affect only ceramide levels in LDL because the enzyme does not act on SM in VLDL particles. To test this, the lipoprotein fraction(s) with elevated ceramide concentration was identified. Lipoproteins were isolated from fresh plasma obtained from 3 of the 11 subjects in the study and ceramide in VLDL, LDL, and HDL was quantified. The levels of ceramide in HDL were not affected by LPS administration. Up to twofold increases in ceramide levels were found in both, VLDL and LDL, fractions (Fig. 3) in all three participants. Surprisingly, the changes in VLDL appeared earlier than the changes in LDL: maximum increases in VLDL were found at 6 h, while the changes in LDL occurred at 24 h after LPS administration. Such pattern implies that early activation of de novo synthesis of ceramide in the liver may contribute to the increases in VLDL ceramide, while the delayed elevation in LDL ceramide may be a result from a normal metabolism of VLDL to LDL and/or from activation of secreted SMase. Furthermore, the differential increases of
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Fig. 2. LPS administration in humans increases the concentration of ceramide in the plasma. The data for each individual (total of 7 subjects shown) are represented with specific hatch pattern or shade. The volunteers were injected i.v. with saline or LPS (20 U per kg. b.w.) in two separate randomized studies separated by 10–20 days of a washout period. Blood was collected at the indicated times after LPS and saline injection. The levels of ceramide in the plasma were measured by TLC/HLPC method and the values at different times after LPS were normalized for the values at the respective time after the saline injection. Saline injection alone did not cause significant changes but there were individual-to-individual differences in the basal levels of ceramide. To account for this, the data are presented as a fold change over the value at time ‘‘0.’’ Based on the different patterns of response to LPS, the subjects were grouped into two groups presented in panels A and B. The ceramide assays were performed in duplicate and the standard deviation was less than 10%. *, p < 0:05; **, p < 0:005.
ceramide in VLDL and LDL fractions may contribute to the appearance of two different patterns of LPS response in regard to the total plasma ceramide levels. LPS induces elevation in serum ceramide in mouse model
Fig. 3. LPS-induces increases in VLDL and LDL ceramide content. Fresh plasma from 3 additional volunteers was used to isolate lipoprotein fractions. Treatments were conducted as described in the legend for Fig. 2. The levels of ceramide were determined by TLC/HPLC method. The data were normalized per the amount of protein present in the lipoproteins, represent average SD, for three ceramide assays, and are representative for the three subjects tested. *, p < 0:05.
The mechanism(s) involved in the elevation of serum ceramide by LPS were tested in a mouse model further. Due to the differences in lipoprotein profiles of humans and mice, two mouse strains were used: C57/Bl6 and LDL receptor (LDLR) knockout mice [24]. The former have very low levels of LDL in the serum. In contrast, the latter lack LDL receptor, they have substantially increased LDL level in the circulation, and resemble lipoprotein profile in humans. LPS was administered i.p. and serum was collected 20 h after the injection. In the C57/Bl6 mice, there was a moderate increase in ceramide levels in total serum that reached 35% over the value for
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Table 2 Elevation in serum ceramide levels in different mouse models
C57/BL6 LDLRKO
Cer DHCer Cer DHCer
Control
LPS
3.49 1.15 ND 7.92 1.51 1.67 0.60
4.74 0.22 ND 12.41 2.16 1.65 0.10
Mice were injected i.p. with LPS and sacrificed after 20 h. Data are expressed as nmol/ml serum and are average SD (n ¼ 3 animals per group). Animal-to-animal deviations are shown.
saline-injected mice (Table 2). In the LDLR knockout mice, the basal and LPS-induced levels were significantly higher than in C57/Bl6, reflecting the higher number of LDL particles. Moreover, the LPS-induced increases were somewhat higher, reaching 60% of the control value.
SPT at 6 h after the injection (Fig. 4). Later time points (20 h post-LPS) showed no differences in SPT activity (not shown). Simultaneous to the activation of SPT, LPS administration also led to increase in S-SMase activity of (Fig. 5, panel A), thus suggesting that both enzymes may contribute for elevating serum ceramide levels. To distinguish between these two possibilities, ASMKO mice were used. These mice lack the gene for ASMase, which encodes the lysosomal sphingomyelinase (L-SMase), as well as the S-SMase. At the same time LPS-induced cytokine secretion including that of TNFa and IL-6 is normal [25]. If LPS administration does not elevate serum ceramide in ASMKO ()/)) mice, then S-SMase is
LPS administration to mice activates both, SPT activity and S-SMase activity Data from earlier studies suggest two possible mechanisms for the elevation of VLDL and LDL ceramide during APR: (i) Stimulation of SPT and de novo synthesis in liver as proposed in hamsters [6,7] and (ii) An increased conversion of LDL SM to ceramide suggested by the increased activity of SSMase in serum of LPS-injected mice [11]. To test the role of each pathway, the activity of SPT in isolated liver microsomes and that of S-SMase in serum were measured. LPS administration led to significant increase in the specific activity of
Fig. 4. Activation of serine–palmitoyl transferase activity in liver microsomes of mice treated with saline and LPS. Mice (n ¼ 5) were injected with LPS (5.8 mg/kg b.w.) or with saline and sacrificed 6 h latter. Livers were excised, equal parts were pooled together, and microsomes were isolated at 105,000g. serine–palmitoyl transferase activity was analyzed in vitro using Palmitoyl CoA and 3 H-labeled L -serine as substrates. The enzymatic product, 3-keto-sphinganine, was separated from the radiolabeled substrate by phase partitioning and measured by scintillation counter. The specific activity was expressed as pmol/mg. Pr/h. Data are average SD (n ¼ 3) and are representative of 2 independent analyses. *, p < 0:05.
Fig. 5. Administration of LPS to ASMKO ()/)) mice causes increases in serum ceramide levels in the absence of S-SMase activity. Male ASMKO (+/+) and ASMKO ()/)) were injected with LPS or saline as described in the legend for Fig. 4. S-SMase activity and ceramide levels in serum were quantified as described in Materials and methods. The data are average SD and animal-to-animal deviations are shown (n ¼ 3 animals per group). *, p < 0:05.
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likely the only enzyme involved. In turn, if the increases in serum ceramide levels are similar in ASMKO ()/)) and ASMKO (+/+) mice, then de novo synthesis of ceramide in the liver is also involved. This result however does not eliminate the role of SSMase completely because ASMKO mice are in C57Bl6 background, and the substrate for S-SMase, LDL, is rapidly cleared from the circulation, thus preventing the detection of differences in ceramide in LDL. Having in mind these limitations, ASMKO ()/)) and litter-matched ASMKO (+/+) mice were injected with LPS or saline. As expected, LPS administration induced activation of S-SMase and elevation of serum ceramide in ASMKO (+/+) mice. In contrast, ASMKO ()/)) had no detectable S-SMase activity in neither saline- nor LPS-injected mice, thus confirming that ASMase is the only secreted, LPS-activated, SMase activity (Fig. 5, panel A). In spite of the lack of SSMase, LPS administration led to elevation in serum ceramide, although the basal levels were somewhat lower than in ASMKO (+/+) mice (Fig. 5, panel B). This indicates that activation of de novo synthesis of ceramide in the liver contributes to the increases in serum ceramide during inflammation.
Discussion This manuscript presents evidence that APR in humans and mice is associated with increases in the mass of ceramide in plasma. Using a human model and mass analyses, our experiments confirm and extend the results obtained earlier in a hamster model (15, 16). The major carriers of ceramide in the plasma are VLDL and LDL; respectively, the LPS administration increased exclusively VLDL and LDL ceramide content. These increases are likely to affect LDL functions in a couple of different ways. One possibility is that elevation of LDL ceramide content may affect the biophysical properties of the particles. Indeed, treatment of gigantic unilamellar liposomes [26] and LDL [9] with sphingomyelinase generates excess ceramide that self-aggregates in ceramide-enriched domains, leading to the aggregation and fusion of the particles. In turn, aggregation of LDL particles may contribute to the development of atherosclerotic plaques. Second, the up-take of ceramide-enriched LDL by the cells of the vasculature may lead to accumulation of ceramide inside the cells. Such accumulation may have an effect on cell functions similar to that mediated by agonist-induced activation of intracellular sphingomyelinases. For example, the up-take of LDL with elevated ceramide content by endothelial cells causes accumulation of ceramide and increases the incidence of apoptosis [17]. Interestingly, increased rate of apoptosis in microvascular endothelium underlies the loss of blood pressure and vascular
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tone during severe endotoxic shock. In addition, the exogenously delivered ceramide may be converted to sphingosine [17] and sphingosine-phosphate, potent proinflammatory second messenger molecules that are implicated in the regulation of adhesion molecule expression [27]. Finally, a third possibility is that by binding to the different LDL-binding receptors, ceramide-rich LDL particles deliver excess ceramide to the plasma membranes, particularly to the lipid rafts. Notably, the formation of ceramide-rich lipid rafts has been suggested to play a role in host response to infection [28]. This manuscript also provides novel evidence for the role of liver SPT during inflammation. Activation of SPT has been correlated with increased production of ceramide inside the cells and induction of apoptosis or cell growth arrest [29]. In lower eukaryotes, for example, SPT is involved in the regulation of heat stress response and yeast strains with mutation in LCB1, one of the two genes encoding SPT, lack the ability to adapt to elevation in temperature [30]. In mammalian cells, activation of SPT plays a role in mediating the responses to different inducers or regulators of cellular apoptosis and growth arrest [31,32]. Data began to accumulate that in the liver, SPT may play an additional role, namely to regulate the rate of ceramide secretion in the circulation. The evidence for such a role can be summarized as follows: (i) In Syrian hamster model, activation of SPT by LPS has been described and correlated to the increases in the secretion of ceramide in plasma lipoproteins. (ii) The data from ASMKO ()/)) mice presented in this manuscript provide additional evidence that increases in serum ceramide levels are mediated via activation of SPT, rather than solely due to activation of SSMase; (iii) SPT is the rate-limiting step in de novo ceramide synthesis and secretion in liver, and activation of SPT in isolated primary hepatocytes causes increases in ceramide level in VLDL [8], while inhibition of de novo synthesis of ceramide by Fumonisin B1 prevents its secretion by hepatocytes. The mechanism for activation of SPT during APR is not completely understood. LPS administration in hamster significantly increases the level of SPT mRNA in the liver in correlation with the increases in its activity [6]. Similar changes are reported in response to cytokines, such as IL-1b, as well as UV irradiation. These observations suggest that transcriptional regulation may be a common mechanism for activation of SPT in proinflammatory conditions; however, the transcription factors involved are not known. The physiological function of regulated ceramide secretion by the liver is unclear. It is possible that the ability to secrete ceramide is a unique feature of that organ related to its role in lipoprotein synthesis and secretion. Stimulation of ceramide release during inflammation may be part of the mechanism by which liver contributes to the dysfunction of vascular endo-
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thelium during APR to infections. Thus, it is possible that in addition to its role of intracellular lipid mediator, ceramide acts as a systemic mediator of inflammation. Alternatively, the regulated secretion of ceramide could be a mechanism for ceramide removal and cell protection during systemic stress.
Acknowledgments This work was supported partly by research award from the American Federation for Aging Research (to M.N.-K) and a Grant-in-Aid from the American Heart Association (Ohio Valley affiliate (to M.N.-K), as well as by the Department of Veterans Affairs Merit Award Program (to S.I.S.) and by M01RR02602 NIH Grant for the University of Kentucky General Clinical Research Center (to S.I.S.). We greatly appreciate the comments of the Cardiovascular Research Group.
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