Advances in Biological Chemistry, 2011, 1, 35-48
doi:10.4236/abc.2011.13006 Published Online November 2011 (
Published Online November 2011 in SciRes.
Alteration of fatty acid molecular species in ceramide and
glucosylceramide under heat stress and expression of
sphingolipid-related genes
Ken-ichi Nagai1, Nobuyoshi Takahashi2, Toshiko Moue3, Yukio Niimura3*
1Department of Clinical Laboratory Science, Faculty of Medical Technology, Teikyo University, Tokyo, Japan;
2Department of Radiological Technology, Faculty of Medical Technology, Teikyo University, Tokyo, Japan;
3Research Center of Biomedical Analysis and Radioisotope, Faculty of Medicine, Teikyo University, Tokyo, Japan.
Email: *
Received 15 September 2011; revised 21 October 2011; accepted 29 October 2011.
Physical stresses such as high temperature or hyper-
osmosis are known causes of intracellular ceramide
(Cer) accumulation in mammalian epithelial cells;
these stresses also result in the activation of the biosy-
ntheses of glucosylceramide (GlcCer) or galactosyl-
ceramide via ceramide glycosylation. We confirmed
that intracellular Cer and GlcCer increased in mouse
fibroblast Mop 8 cells under conditions of heat stress.
When molecular species of Cer, GlcCer and sphingo-
myelin (SM) were analyzed by matrix assisted laser
desorption ionization time of flight mass spectrome-
try (MALDI-TOF MS), the molecular ion peaks of
Cer (d18:1 - C16:0, Na+) and Cer (d18:1 - C22:0, Na+)
increased under heat stress compared with those of
Cer (d18:1 - C24:1, Na+) and Cer (d18:1 - C24:0, Na+).
GlcCer and SM demonstrated the wide spectra of
fatty acyl chains compared with that of Cer. The ratio
of GlcCer consisted of hydroxy fatty acid to that con-
sisted of non-hydroxy fatty acid increased 2-5-fold in
heat stressed cells. Cer metabolism-related genes, se-
rine palmitoyltransferase (Spt), ceramide synthase-1,
-2, -4, -5 and -6 (CerS1, -2, -4, -5 and -6), neutral
sphingomyelinase-1 and -2 (nSMase1 and nS-Mase2),
sphingomyelin synthase-1 (SgmS1), and ceramide glu-
cosyltransferase (GlcT), were activated after 16 h un-
der heat stress at 42˚C. Activation of Sg-mS1 and
GlcT genes played a role as Cer scavengers in the
decrease of intracellular Cer levels. Activation of Cer-
S5 and/or CerS6 gene may contribute to the accu-
mulation of Cer species of (d18:1 - C16:0) under heat
Keywords: Ceramide; Glucosylceramide; Heat Stress;
Fatty Acids; Ceramide Synthases
Ceramide (Cer) plays the role of a lipid second messen-
ger in cell signaling transductions [1-5] such as cell
growth, differentiation [6], senescence [7], necrosis [8],
proliferation [9], and apoptosis [10]. Changes in Cer
metabolism are also implicated in the etiology of human
diseases, such as neurological disorders, cancer, infec-
tious diseases, and Wilson’s disease [11,12]. The accu-
mulation of Cer in response to heat-shock stress has
been shown in HL-60 [13,14] and NIH WT-3T3 cells [15]
and is dependent on the activities of both serine palmi-
toyltransferase (Spt) and ceramide synthase (CerS) [16].
The generation of Cer may result from de novo synthesis
and/or sphingomyeline (SM) degradation by acid sphin-
gomyelinase (aSMase) or neutral sphingomyelinase (nS-
Mase). The nSMase which hydrolyzes SM to generate
Cer plays a critical role in stress responses including
apoptosis [17]. Cer is an important initial precursor in
sphingoglycolipid metabolism. Cer glycosylation is in-
volved in the regulation of Cer content in B16 melanoma
cells [18], and glucosylceramide (GlcCer) synthesis pro-
tects against Cer-induced stress in keratinocytes [19]. In
previous studies we demonstrated that hyperosmotic stress
to Madin-Darby canine kidney (MDCK) cells increased
Cer content, and the mRNA level of Cer galactosyl-
transferase (GalT) was upregulated resulting in galacto-
sylceramide (GalCer) accumulation [20]. Hyperosmotic
stress also affected the syntheses of more complex gly-
cosphingolipids such as sulfoglycolipids by the mito-
gen-activated protein kinase (MAPK) signaling pathway
[21]. In our recent study [22], heat-stressed MDCK cells
had an increased content of Cer, and the de novo synthe-
sis from serine and both GlcCer and GalCer syntheses
from Cer were shown to increase by metabolic labeling.
The gene expression of Cer glucosyltransferase (GlcT)
and GalT also increased significantly under heat stress.
K.-I. Nagai et al. / Advances in Biological Chemistry 1 (2011) 35-48
In this study we examined Cer levels, molecular species
of Cer, GlcCer and SM, and expression of sphingoli-
pid-related genes in mouse fibroblasts subjected to heat
2.1. Reagents and Standards
Protein concentrations were determined with Quick Start
Bradford Dye Reagent (Bio-Rad, Hercules, CA, USA).
GlcCer and lactosylceramide (LacCer) were prepared in
our laboratory from horse kidney and human kidney,
respectively. GalCer from bovine brain, phosphatidyl
se-rine (PS), phosphatidyl choline (PC), phosphatidyl
ethanolamine (PE) and SM were obtained from Sigma-
Aldrich Co. (St. Louis, MO, USA). Cer from bovine
brain was purchased from Funakoshi Co. (Tokyo, Japan).
Thin layer chromatography (TLC) was performed on
Silica gel 60 high-performance TLC (HPTLC) plates
(Merck, Darmstadt, Germany). Orcinol reagent was used
for detection of glycolipids and primuline reagent was
used for detection of lipids.
2.2. Cell Culture
Mop-8 cells [23] with a hybrid transcription unit com-
posed of the SV40 early promoter fused to the early re-
gion of Polyoma virus were purchased from American
Type Culture Collection (ATCC) and cultured in Dul-
becco’s modified Eagle’s medium (D-MEM, Nissui Pha-
rmaceutical Co., Tokyo, Japan) containing 10% fetal bo-
vine serum (FBS) (Invitrogen, Carlsbad, CA, USA).
Heat stress was introduced at 40˚C or 42˚C.
2.3. Metabolic Labeling of Cer and Glycolipids
14C(U)-L-serine (24.7 kBq/ml; Moravek Biochemicals
Inc., Brea, CA, USA) was incorporated into Cer for 16 h
under various conditions. Lipids were extracted and then
partitioned with a Folch solvent system containing 0.88%
potassium chloride. An aliquot of the lower phase was
analyzed by TLC using stepwise development with two
solvents: the first was with chloroform/methanol/acetic
acid (9:1:1, v/v) up to the top of the plate, and the second
was with chloroform/methanol/ammonia (65:35:7.5, v/v)
up to 60% of the top of the plate. Following TLC, an
imaging analyzer (FLA-7000, Fujifilm, Tokyo, Japan)
was used to determine radioactivity incorporated into the
lipids. Cer, PE, PS, PC, and SM used as standards for
TLC were detected by spraying the primuline reagent.
Labeling with [1-14C]-D-galactose (Moravek Biochemi-
cals Inc.) was performed under the directed conditions.
Cells were cultured for 5 h with 14C-galactose (37 kBq/
ml), after which lipids were extracted twice with chlo-
roform/methanol (2:1 and 1:2, v/v). Cells were then
treated with alkaline methanol. After neutralization and
desalting, the neutral glycolipid fraction was obtained
using a DEAE-Sephadex® A-25 (Pharmacia Fine Chemi-
cals AB, Uppsala, Sweden) column. Neutral glycolipids
were two-dimensionally (2D) developed on HPTLC
plates using chloroform/methanol/water (60:25:4, v/v)
and 2-propanol/ammonium hydroxide/methylacetate/water
(15:2:1:3, v/v) [20-22,24]. Total radioactivity incorpo-
rated into the glycolipids was measured using a liquid
scintillation counter (Packard Instrument Co. Inc., Do-
wners Grove, IL, USA).
2.4. Preparation of Glycolipids and TLC Analysis
Non-radioactive glycolipids were also prepared from
cells by chloroform/methanol extraction, mild alkali treat-
ment, DEAE-Sephadex® A-25 column chromatography,
and TLC as mentioned previously. The glycolipids on
TLC were visualized by spraying the plate with orcinol-
H2SO4 reagent and heating for several minutes at 120˚C.
2.5. Mass Spectrometric Analysis
Matrix-assisted laser desorption ionization time of flight
mass spectrometry (MALDI-TOF MS) for lipid samples
was carried out on an AXIMA Performance mass spec-
trometer (Shimadzu Biotech, Manchester, UK) using 2,
5-dihidroxy benzoic acid (DHB, Bruker Daltonics, Bre-
men, Germany) saturated with sodium chloride as a ma-
trix. Angiotensin II (Shimadzu GLC Ltd.; MH+ of
1046.54), C16 Cer (Funakoshi; MH+ of 537.90) and α-
cyano-4-hydroxycinnamic acid (Shimadzu GLC Ltd.;
MH+ of 190.05) were used for mass calibration. Lipid
solution (0.5 μL) was mixed with a matrix solution (0.5
μL) on the surface of a stainless steel MALDI-TOF plate.
All spectra were acquired in the positive ion reflectron
mode using a mass spectrometer equipped with a nitro-
gen UV laser (337 nm). All mass spectrometric data
were acquired and analyzed using MALDI-MS software.
Typically, 4000 laser shots were acquired for each MS
spectrum, and spectra were obtained generally at a laser
power of 80 in an attempt to maximize resolution and
peak intensity, and analyzed using MALDI-MS software.
Helium gas was used for high-energy collision-induced
dissociation (CID) fragmentation for MS/MS analysis.
2.6. Quantification of MRNA by Real-Time
Polymerase Chain Reaction (PCR)
Total RNA was isolated from cells using Trizol reagent
(Invitrogen), according to the manufacturer’s instruct-
tions. A Transcriptor High Fidelity cDNA Synthesis Kit
(Roche Diagnostics GmbH, Mannheim, Germany) was
used for reverse transcription of mRNA. The product
was appropriately diluted with water and used as a tem-
plate. ProbeFinder (Roche Diagnostics GmbH) for mouse
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K.-I. Nagai et al. / Advances in Biological Chemistry 1 (2011) 35-48
Copyright © 2011 SciRes.
mRNA was used to design the optimal real-time PCR
assay. For the optimal real-time PCR assay of mouse Spt,
long chain base subunit 1 (Sptlc1) mRNA, Universal Pro-
beLibrary probe #67 (no. 04688660001, Roche Diagnos-
tics) and a primer set (5’-ggtgctggtggagatggt-3’ and 5’-
ggattccttccaaaataagatgg-3’) were used. The 68-base pair
(bp) amplicon was detected by agarose gel electrophoresis.
Additional Universal ProbeLibrary (UPL) probes and pri-
mers were used for real-time PCR assays of mouse Sptlc2,
CerS1 - CerS6, nSMase1, nSMase2, SgmS1, GlcT and
HSP70 mRNAs as listed in Table 1.
The mouse glyceraldehyde-3-phosphate dehydrogena-
se (Gapdh) gene was used as the UPL reference gene. A
PCR mixture (20 μL/well) was prepared on 96-well pla-
tes with LightCycler® 480 Probe Master Reagent. The re-
action was carried out with a LightCycler® 480 (Roche
Diagnostics GmbH). PCR conditions were as follows:
pre-incubation (95˚C·5 min), amplification (95˚C·10 s,
4.4˚C/s), (60˚C·25 s, 2.2˚C/s), and (72˚C·1 s, 4.4˚C /s),
for 45 - 65 cycles. Data (n = 6) were standardized by
ratio using the expression of mouse Gapdh as an internal
2.7. Statistical Analysis
Statistical significance was evaluated without using one-
way ANOVA by unpaired t tests as applicable; P < 0.05
was considered statistically significant.
3.1. Accumulation of GlcCer in Mop8 Cells
under Heat Stress
After being subjected to heat stress at 42˚C for 15 h,
glycolipids of mouse fibroblast Mop8 cells were meta-
bolically labeled with 14C-galactose for 5 h. The neutral
glycolipids from Mop8 cells were separated by 2D-
HPTLC analysis using a two-solvent system. The plates
were analyzed by an imaging analyzer or spraying orci-
nol reagent. Figures 1(a) and (b) show the glycolipid
profiles of Mop8 cells in a control culture and heat-
stressed culture at 42˚C, respectively. GlcCer and Lac-
Cer of Mop8 cells co-migrated with their authentic refe-
rences. The 14C-incorporation into GlcCer in the heat-
stressed culture increased 20% compared to the control
at 37˚C (Figure 1(c)), while LacCer decreased to 53%
(Figure 1(d)).
Table 1. Probes and primers for real-time PCR.
Enzyme Probe # Gene Primers Sequence Amplicon (bp)
Left 5’-ggtgctggtggagatggt-3’
67 Sptlc1
(subunit1) Right 5’-ggattccttccaaaataagatgg-3’ 68
Left 5’-tcggtgcttcaggaggatac-3’
Serine palmitoyltransferase
17 Sptlc2
(subunit2) Right 5’-gagaatgtgtgcgcaggtag-3’ 66
Left 5’-ctcattgcctcttcctacgc-3’
Ceramide synthase 1 4 CerS1 Right 5’-cagctgcacatcgctgac-3’ 84
Left 5’-agaagtgggaaacggagtagc-3’
Ceramide synthase 2 50 CerS2 Right 5’-ttcccaccagaagtagtcatacaa-3’ 95
Left 5’-ggcgatttacattttacttgctg-3’
Ceramide synthase 3 19 CerS3 Right 5’-ggtcatatgcccatggtttg-3’ 75
Left 5’-gcctgcatcttgctttctg-3’
Ceramide synthase 4 67 CerS4 Right 5’-ctgccacagccactcactc-3’ 62
Left 5’-catgccatctggtcctacct-3’
Ceramide synthase 5 38 CerS5 Right 5’-gcggtcatccttagacacct-3’ 78
Left 5’-ggagctgtcattttattggtcttt-3’
Ceramide synthase 6 48 CerS6 Right 5’-ggaacataatgccgaagtcc-3’ 77
Left 5’-tcctggaggaggtgtgga-3’
Neutral sphingomyelinase 1 15 nSMase1 Right 5’-agaggccactgcctatcatc-3’ 111
Left 5’-tctacctcctcgaccagcac-3’
Neutral sphingomyelinase 2 17 nSMase2 Right 5’-tgctgctccagtttgtcatc-3’ 110
Left 5’-ggcatgcatttcaactgttc-3’
Sphingomyelin synthase 1 48 SgmS1 Right 5’-gagcttcattattctccgcact-3’ 72
Left 5’-gtttcaatccagaatgatcaggt-3’
UDP-glucose ceramide
glucosyltransferase 4 GlcT
Right 5’-aagcattctgaaattggctca-3’ 87
Left 5’-aaaatactggataaatgcaatgagg-3’
Heat shock protein 70 20 HSP70 Right 5’-ttatgatcaaactcatctttctcagc-3’ 80
K.-I. Nagai et al. / Advances in Biological Chemistry 1 (2011) 35-48
Figure 1. Change of glycolipids profile in Mop8 cells under heat stress. Confluent cells were cultured at 37˚C and
42˚C for 15 h. Cells were then labeled with 14C-galactose (37 kBq/ml) at 37˚C and 42˚C for 5 h. Neutral glycolipids
were prepared as described in the MATERIALS AND METHODS section and developed by 2D HPTLC with
chloroform /methanol/water (60:25:4, v/v) in the vertical direction and 2-propanol/ammonium hydroxide/methyl
acetate/water (15:2:1:3, v/v) in the horizontal direction. The 14C-incorporation into neutral glycolipids was analyzed
with the radio imaging. Reference glycolipids were visualized with spraying the orcinol reagent. (a) control culture
at 37˚C, a; GlcCer (horse kidney), b; GalCer (bovine brain), c; LacCer (human kidney), (b) heat stress at 42˚C, (c)
incorporation into GlcCer, (d) incorporation into LacCer. *Significance difference, <0.05 (n = 4).
3.2. Cer Increment and Phospholipid
Metabolism under Heat Stress
Cer and phospholipids extracted from cells were ana-
lyzed by HPTLC using a two-step development process
with two solvent systems. Heat stress at 42˚C for 16 h on
Mop8 cells increased the incorporation of 14C-serine into
Cer by 17% compared to the control culture at 37˚C,
while incorporation into phospholipids (PE, PS, and SM)
decreased significantly under heat stress (Figure 2(a),
(b)). Cer content was also determined by spraying pri-
muline reagent. An increase of 23% was observed under
heat stress (Figure 2(c)).
3.3. Analysis of Sphingolipid Molecular Species
Cer prepared from HPTLC was analyzed by MALDI-
TOF MS. Cer from unstressed cells showed 4 ion peaks
at m/z 560.23 (peak height: 100), m/z 644.30 (peak
height: 13), m/z 670.30 (peak height: 80) and m/z 672.30
(peak height: 46), whereas that from heat stressed cells
showed the ion peaks at m/z 560.17 (peak height: 100),
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K.-I. Nagai et al. / Advances in Biological Chemistry 1 (2011) 35-48 39
Figure 2. Changes of Cer and phospholipids metabolism under heat stress. Confluent cells were cultured
with 14C-serine (37 kBq/ml) at 37˚C and 42˚C for 16 h. Total lipids prepared as described in the MATE-
RIALS AND METHODS section were separated by HPTLC with chloroform/methanol/acetic acid (9:1:1,
v/v) in the first run, the plate was dried and then re-developed with chloroform/methanol/acetic acid/ water
(25:15:4:2, v/v) until 65% of the solvent top of the first run. (a) representative TLC imaging patterns of
lipids, (b) 14C-incorporation into lipids, (c) Cer content determined by primuline reagent. *Significance
difference, <0.05 (n = 4).
m/z 644.23 (peak height: 15), m/z 670.22 (peak height:
26) and m/z 672.26 (peak height: 14) (Figures 3(a) and
(b), Table 2).
Cer at m/z 560 corresponds to a sodiated molecular ion
composed of d18:1 as a long chain base and C16:0 as a
fatty acid. MS/MS analysis of m/z 560 with high-energy
CID fragmentation showed typical fragments m/z 264
and m/z 376 derived from d18:1, and m/z 304 from C16:0
(Figure 3(c)) [25]. Ions (m/z 390, 404, 418, 432, 446,
460, 474, 488, 516 and 544) with a difference of 14 Da
(-CH2-) between ions were observed between 560 and
376, indicating the ions derived from C16:0 and d18: 1.
Cer at m/z 670 and m/z 672 corresponds to sodiated mo-
lecular ions composed of d18:1 as a long chain base and
C24:1 and C24:0 as fatty acids, respectively. Further, the
analysis of m/z 670 - 672 with CID fragmentation re-
vealed m/z 264 (not shown) and m/z 376 derived from
d18:1, and m/z 416 from C24:0, (Figure 3(d)) [25].
While it was hard to detect m/z 414 derived from C24:1,
molecular ion m/z 670 was speculated as d18:1 - C24:1
from the metabolic relationship. Ion peak of Cer at m/z
644 was tentatively assigned as a sodiated molecular ion
composed of d18:1 as a long chain base and C22:0 as a
fatty acid, and the ratio of the peak height to that of m/z
670 (d18:1 - C24:1, Cer) and m/z 672 (d18:1 - C24:0, Cer)
was higher in heat stressed cells than in the control cells
(Figure 3(a) and (b)). These results suggested that d18:1 -
C16:0 and d18:1 - C22:0 Cer species increased
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K.-I. Nagai et al. / Advances in Biological Chemistry 1 (2011) 35-48
Figure 3. Analysis of Cer with MALDI-TOF MS. (a) Cer from control cells, (b) Cer from heat-stressed cells at 42˚C, (c) analysis of
m/z 560 with CID, (d) analysis of m/z 670 - 672 with CID. x; unidentified peak.
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K.-I. Nagai et al. / Advances in Biological Chemistry 1 (2011) 35-48
Copyright © 2011 SciRes.
Table 2. Molecular ion species in Cer and GlcCer.
Cer GlcCer
control heat stress control heat stress
molecular species in Cer moiety m/z peak height m/z peak heightm/z peak height m/z peak height
(d18:1 - C16:0, Na+) 560.23 100.0 560.17 100.0 722.60 100.0 722.61 100.0
(d18:1 - C16:0 (OH), Na+) - - - - 738.58 61.0 738.57 139.0
(d18:1 - C18:0, Na+) - - - - 750.64 5.4 750.64 7.8
(d18:1 - C18:0 (OH), Na+) - - - - 766.61 3.6 766.60 8.1
(d18:1 - C20:0, Na+) - - - - 778.67 4.2 778.66 5.0
(d18:1 - C20:0 (OH), Na+) - - - - 794.65 2.8 794.63 7.2
(d18:1 - C22:0, Na+) 644.30 13.0 644.23 15.0 806.70 6.0 806.70 7.8
(d18:1 - C22:0 (OH), Na+) - - - - 822.68 4.2 822.67 11.9
(d18:1 - C24:1, Na+) 670.30 80.0 670.22 26.0 832.71 7.2 832.71 18.3
(d18:1 - C24:0, Na+) 672.30 46.0 672.26 14.0 834.72 9.2 834.72 17.5
(d18:1 - C24:1 (OH), Na+) - - - - 848.69 5.6 848.68 27.8
(d18:1 - C24:0 (OH), Na+) - - - - 850.70 7.2 850.69 25.0
more than d18:1 - C24:1 Cer and d18:1 - C24:0 Cer in
heat stressed cells. The changes of molecular species of
GlcCer and SM under heat stress were also analyzed.
Molecular ion species of GlcCer showed a wide spec-
trum of fatty acyl chain compared with that of Cer. So-
diated molecular ions consisting of a hexose, d18:1 as a
long chain base and a fatty acid corresponding to C16:0,
C16:0 (OH), C18:0, C18:0 (OH), C20:0, C20:0 (OH),
C22:0, C22:0 (OH), C24:0, C24:0(OH), C24:1 and C24:
1 (OH), respectively, were detected in both control and
heat stress-ed cells (Figures 4 (a) and (b)).
The MS/MS analysis of m/z 722.60 with CID frag-
mentation revealed m/z 304.15 derived from a fatty
acid C16:0 and m/z 539.72 from d18: 1 as a long chain
base (Figure 4). The relative peak height of each mo-
lecular ion to (d18:1 - C16:0, Na+) was obtained (Ta-
ble 2). The relative peak ratio of each GlcCer con-
sisting of hydroxy fatty acid to that consisting of
non-hydroxy fatty acid increased 2-5-fold in heat stre-
ssed cells compared with that in control cells. Whe-
reas, the analysis of SM molecular species demon-
strated several molecular ions identified as indicated
in Figure 4(d) between m/z 700 and m/z 860 with a
small difference in control and heat stressed cells
(Figures 4(d) and (e)). The major ion peak was m/z
725(d18:1-C16:0, Na+).
3.4. Activation of Cer-Related Genes and HSP70
Gene under Heat Stress
We investigated the effects of heat stress on selected
genes. Spt, known as a first-step enzyme in de novo syn-
thesis of Cer, is composed of two subunits: Sptlc1 and
Sptlc2. We found the activation of both Sptlc1 and
Sptlc2 genes (at 42˚C for 16 h) to be 2.67-fold and 2.84-
fold, respectively (Figure 5(a)). In a time-dependent
experiment, the Sptlc1 gene increased by 1.64-fold at 8h
and 2.67-fold at 16h (Figure 5(b)). Expression of 6 CerS
genes (CerS1-CerS6) known from mouse tissues, was
investigated under heat stress. The CerS genes were sig-
nificantly activated at 42˚C to 23.82-fold (CerS1), 2.70-
fold (CerS2), 2.17-fold (CerS4), 1.92-fold (CerS5) and
2.41-fold (CerS6), while CerS3 was not significantly
activated at 42˚C. Only CerS6 gene was activated to
1.24-fold at 40˚C (Figures 6(a)-(f)). The expression lev-
els (10–3 or 10–2) of CerS4-CerS6 to the reference gene
were much higher than those (10–7) of CerS1-CeS3.
The nSMase1, nSMase2, and SgmS1 genes were also
activated to 2.17-fold, 3.02-fold, and 1.65-fold, respec-
tively, at 42˚C (Figur es 7(a)-(c)) but not at 40˚C. The GlcT
gene was significantly activated to 1.22-fold at 40˚C and
1.60-fold at 42˚C (Figure 7(d)). The GlcT gene was acti-
vated to 1.41-fold at 8h and 1.60-fold at 16 h (Figure 7(e)).
The HSP70 gene as a marker of heat stress was also en-
hanced to 3.44-fold at 42˚C, but not at 40˚C (Figur e 7(f)).
K.-I. Nagai et al. / Advances in Biological Chemistry 1 (2011) 35-48
Figure 4. Analysis of GlcCer and SM with MALDI-TOF MS. (a) GlcCer from control cells, (b) GlcCer from heat-stressed cells at
42˚C, (c) analysis of m/z 722 with CID, (d) SM from control cells, (e) SM from heat-stressed cells at 42˚C.
Figure 5. Activation of Spt gene in Mop8 cells under heat stress. Confluent Mop8 cells were cultured at 37, 40 and 42˚C for 16 h.
Total RNAs were prepared and used for quantitative RT-PCR as described in the MATERIALS AND METHODS section. (a) Sptlc1
gene and Sptlc2 gene, (b) time-dependent activation of Sptlc1 gene.
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K.-I. Nagai et al. / Advances in Biological Chemistry 1 (2011) 35-48
Copyright © 2011 SciRes.
Figure 6. Expression of CerS genes under heat stress. (a) CerS1, (b) CerS2, (c) CerS3, (d) CerS4, (e) CerS5, (f) CerS6. Longitudinal
axis represents the ratio of a target gene to Gapdh as a reference gene. *Significance difference, <0.05 (n = 6).
4. DISCUSSION which are the first glycosylation steps of sphingolipid
metabolism in kidney cells, also increased with the acti-
vation of both GlcT and GalT genes. Thus, we speculated
that accumulation of Cer under heat stress might be a
trigger to activate the synthesis of monohexosylcera-
mides (GalCer and/or GlcCer) to decrease its level and
evade cell apoptosis. In the present study, we confirmed
that the increment of Cer and its subsequent glycosyla-
tion also occurred in mouse fibroblast Mop8 cells under
heat stress.
Cer is a major bioactive lipid in eukaryotic cells [2]. In
addition to its structural significance as a membrane
component, it is believed to be involved in a variety of
cellular functions including the regulation of cell growth,
differentiation, and viability. It is well known that vari-
-ous stresses such as high temperature and ultraviolet
(UV) radiation elevate intracellular Cer levels followed
by cell apoptosis [4,5]. We found that hypertonic stress
elicited the syntheses of both Cer and GalCer in kidney
cells [20]. In our recent study [22], heat stress on MDCK
cells increased Cer content and its de novo synthesis from
serine. Both GlcCer and GalCer syntheses from Cer,
The enzyme genes of Spt, CerS1, CerS2, CerS4, Cer-
S5, CerS6, nSMase1, and nSMase2 relevant to the eleva-
tion of intracellular Cer contents were shown to be acti-
vated under heat stress at 42˚C. On the other hand, the
K.-I. Nagai et al. / Advances in Biological Chemistry 1 (2011) 35-48
message signals of SgmS1 and GlcT corresponding to
lowering the Cer level were also activated. Cer is newly
synthesized at the cytosolic surface of the endoplasmic
reticulum from serine and palmitoyl-CoA, which con-
dense to form 3-ketosphinganine through the action of
Spt [26], followed by a rapid reduction to sphinganine by
3-ketosphinganine reductase. Synthesis of more complex
sphingolipid metabolites such as SM and GlcCer is car-
ried out in the Golgi apparatus [4]. Heatshock-induced
Cer accumulation in mammalian cells is known to take
place during de novo synthesis [16]. The activation of
GlcT and SgmS under heat stress suggests that heat
stress may affect activation of enzymes in the Golgi in
addition to those in the endoplasmic reticulum.
Figure 7. Expression of sphingolipid-related genes and HSP70 gene under heat stress. (a) nSMase1, (b) nSMase2, (c) SgmS1, (d)
GlcT, (e) time-dependent activation of GlcT gene, (f) HSP70. Longitudinal axis represents the ratio of a target gene to Gapdh as a
reference gene. *Significance difference, <0.05 (n = 6).
opyright © 2011 SciRes. ABC
K.-I. Nagai et al. / Advances in Biological Chemistry 1 (2011) 35-48 45
Cer homeostasis is also dependent on de novo synthe-
sis of Cer from sphinganine and acyl CoA, catalyzed by
a family of six CerS (CerS1-6). These enzymes are pro-
ducts different genes and preferentially use different fa-
tty acyl CoA substrates containing fatty acid chains of
different lengths, thereby producing Cer with different
acyl chains [27]. CerS1 is a mammalian homolog of the
yeast longevity assurance gene 1. It has been shown in
yeast that longevity assurance gene members play a role
in the regulation of life span and are also required for
CerS activity. Recent studies also revealed that one of
the mouse homologs of these proteins specifically regu-
lates the synthesis of stearoyl (C18)-containing sphin-
golipids, including C18-Cer [28]. CerS1 is the most stru-
cturally and functionally distinct enzyme among the me-
mbers and the level of CerS1 is specifically controlled
via ubiquitination and proteasome-dependent protein tu-
rnover. Both endogenous and ectopically expressed Cer-
S1 have rapid basal turnover, and diverse stresses in-
cluding chemotherapeutic drugs, UV light, and DTT can
cause increased turnover of CerS1 [29]. In our experi-
ment the highest expression of the CerS1 gene at 42˚C
seemed to reflect the greatest heat stress sensitivity
among the Cer-related genes investigated. However, the
significance of higher response of CerS1 to heat stress is
unclear because GlcCer and SM consisted of C18 fatty
acyl chain corresponding to this gene have been detected
weakly in this cell line, but not in Cer. CerS2 mRNA has
the broadest tissue distribution, and synthesizes Cer
containing mainly C20-C26 fatty acids, with little or no
synthesis of C16- and C18-Cer [30].
The molecular heterogeneity of Cer synthesized in the
epidermis and their possible roles in epidermal perme-
ability barrier functions have also been investigated [31].
The expressions of CerS2, CerS4, CerS5 and CerS6
were enhanced at lower extent than that of CerS1. From
the result that the expression levels of CerS4-CerS6 to
the reference gene were much higher than those of
CerS1-CerS3 in both control and heat-stressed cells, the
enzyme activities of CerS4-CerS6 may greatly affect the
composition of fatty acid moieties of Cer under heat
stress. CerS5 and CerS6 have been known to use C16-
CoA as a substrate, while CerS2 could use C24:0-CoA
and C24:1-CoA [32]. CerS5 and/or CerS6 may contrib-
ute to the Cer (d18:1-C16:0) accumulation under heat
stress in this cell line. CerS2 may also contribute to the
increase of GlcCer containing C24:0 or C24:1 under heat
stress in this cell line. The relationship between the en-
zyme activities of CerS members and the fatty acyl chain
of Cer should be analyzed further. Hydroxy fatty acyl
chains were detected from GlcCer and SM. All CerS
members can synthesize 2-hydroxy fatty acyl-Cer with
specificity for 2-hydroxy-fatty acyl-CoA chain length
[33]. A gene of a fatty acid 2-hydroxylase (FA2H),
which forms 2-hydroxy fatty acids from non-hydroxy
fatty acids, has been characterized [34]. Activation of
FA2H under heat stress may contribute to the enhance-
ment of hydroxyl fatty acid molecular species of GlcCer.
We previously reported the increment of hydroxy fatty
acid species in sulfatide and GalCer under hyperosmotic
stress in kidney cells [35]. Although, little is known
about the significance of hydroxy fatty acid molecular
species of sphingolipids, their hydrophilic properties
increase the hydroxy bonds in the bilayer of the plasma
membrane. Enrichment of Cer with a short chain fatty
acid and GlcCer with hydroxy fatty acyl chains under
heat stress may reduce the hydrophobicity of the lipid
molecule, and increase the membrane fluidity and affect
the properties of the cell membrane to adapt to stress-
A report has suggested that Cer was generated by SM
hydrolysis by heat-shock-activated, magnesium-depen
dent nSMase [36]. Consistent with the report, both nSM-
ase1 and nSMase2 were revealed to be upregulated un-
der heat stress in our experiment. SM hydrolysis in re-
sponse to stress-inducing agents and subsequent Cer
generation are implicated in various cellular responses,
including apoptosis, inflammation, and proliferation,
depending on the nature of the different aSMases or
nSMases. The nSMases are activated by a variety of s-
tress-inducing agents including cytokines, oxidative stre-
ss (H2O2, oxidized lipoproteins), UV radiation, chemo-
therapeutic drugs, amyloid peptides, and lipopolysac-
charides [37]. In mammals, Mg2+-dependent nSMases,
nSMase1, nSMase2, and nSMase3, have been identified.
Among these enzymes, nSMase2 is most studied and has
been implicated in multiple physiological responses in-
cluding cell growth arrest, apoptosis, development, and
inflammation [38]. The nSMase1 knockout mice showed
no abnormality in sphingolipid metabolism or lipid sto-
rage disease [39].The physiologic role of nSMase1 is
still unclear and further elucidation is necessary; how-
ever, nSMase2 has emerged as a major candidate for stre-
ss-induced Cer production and a number of anti-cancer
drugs have been shown to exert effects on nSMase2. In
MCF-7 breast cancer cells, daunorubicin upregulated
cellular nSMase activity and Cer levels [40]. Other re-
ports have also suggested that nSMase2 plays a role in
stress-induced bronchial and lung injury in pulmonary
diseases [41,42]. In mammalian cells the bulk of Cer is
converted to SM by SgmS1 in the lumen of the trans-
Golgi. This enzyme catalyzes the transfer of phospho-
choline from PC to Cer. A second SgmS, SgmS2, resides
at the plasma membrane, but it is unclear whether this
enzyme participates in de novo synthesis of SM [43]. It
could be expected that activation of anabolic SgmS1 and
opyright © 2011 SciRes. ABC
K.-I. Nagai et al. / Advances in Biological Chemistry 1 (2011) 35-48
catabolic nSMase1 and nSMase2 under heat stress cau-
sed the enhancement of SM turnover in our experiment.
In fact, we observed a significant decrease of SM radio-
activity to 81% at 42˚C which coincided with the total
increase of 17% of serine incorporation into Cer or the
increase of 23% of Cer content (Figure 2). SMasein-
duced stress initiates a response in keratinocytes that in-
cludes upregulation of GlcCer synthesis which may pro-
tect against the deleterious effects of excess Cer [19]. In
PDMP (1-phenyl-2-decanoylamino-3-morpholino-1-propa-
nol) treated cells there is a decrease in the amount of
cellular GlcCer and other GlcCer-based glycolipids, whe-
reas the amount of Cer increases [44,45]. The induction
of Cer accumulation by various triggers of Cer genera-
tion elicits the activation of caspase-3. This elevation of
Cer levels also induces cleavage of the death substrate
poly (ADP-ribose) polymerase followed by apoptotic
cell death [46]. The significance of GlcCer synthase in
apoptosis and drug resistance has been investigated
[47,48]. We previously reported that the GlcT gene was
activated in kidney cells under heat stress and the syn-
thesis of GlcCer was enhanced by the introduction of
HSP70 gene into the cells [22]. In the present study, we
confirmed that the GlcT gene in fibroblasts was acti-
vated in a temperature-dependent manner under heat
stress. This activation of GlcT induced an increase of
GlcCer, resulting in a decrease in intracellular Cer. The
salvage of Cer may prevent cell apoptosis. However,
another reason for the accumulation of GlcCer may be
caused of decrease of LacCer synthesis from GlcCer as
shown in the metabolic labeling. Activation of sphingol-
ipid-related genes under heat stress seems to correlate
with the activation of HSP70 gene. The precise mecha-
nism underlying the activation of these genes remains to
be elucidated.
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