Thermally induced changes in lipid composition of raft and non-raft regions of hepatocyte plasma membranes of rainbow trout
School of Life Sciences, Arizona State University, Tempe, AZ, USA
* Author for correspondence (e-mail: john.zehmer{at}utsouthwestern.edu)
Accepted 26 September 2005
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Summary |
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Key words: raft, trout, Oncorhynchus mykiss, lipid composition, cholesterol, fatty acid, poikilothermy
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Introduction |
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During acclimation to elevated temperatures, highly conserved lipid
compositional changes in the PM include a decrease in the number of double
bonds in membrane lipid fatty acids (Hazel
and Williams, 1990; Cossins,
1994
) and an increase in the mole fraction of PM cholesterol
(Robertson and Hazel, 1995
).
These changes have commonly been interpreted as mechanisms for compensation of
membrane fluidity/order, a physical property that is thought to impact the
function of proteins embedded in the membrane
(Cossins, 1994
).
However, these compositional changes are also consistent with the thermal
stabilization of the raft/non-raft phase separation that is thought to give
rise to raft structure. In mammals, rafts are enriched in cholesterol, lipids
with saturated acyl chains (sphingomyelin in particular), and glycolipids
(Fridriksson et al., 1999;
Prinetti et al., 2000
;
Pike et al., 2002
).
Interactions between cholesterol and lipids with saturated acyl chains are
thought to result in the lateral segregation of liquid-ordered (Lo)
phase raft and liquid-disordered (Ld) phase non-raft PM
(Ahmed et al., 1997
).
Sphingomyelin has a particularly strong interaction with cholesterol
(Lund-Katz et al., 1988
;
Ramstedt and Slotte,
1999
).
Since lipid phase behavior is temperature sensitive, it is reasonable to
hypothesize that compositional changes in the PM act to stabilize rafts in the
face of thermal perturbation. The increase in fatty acid saturation and
cholesterol content observed in the total PM of animals acclimated to elevated
temperature are consistent with the stabilization of rafts against thermal
perturbation. Furthermore, we have found that detergent saturation point, an
index of membrane packing strength (and thus raft stability), was conserved in
raft but not in raft-depleted plasma membrane (RDPM) of trout liver PM
(Zehmer and Hazel, 2003).
Curiously, we found raft from warm-acclimated trout to be only slightly more
ordered than RDPM but there was no difference in order between raft and RDPM
from cold-acclimated trout (Zehmer and
Hazel, 2004
). Since, in mammals, raft-RPDM segregation is thought
to be based on a separation of ordered and disordered phases, it is not clear
what maintains the separation in fish membranes.
Therefore, to better understand the regulation of the physical properties of raft and RPDM and the mechanism of the segregation of these domains in poikilotherms acclimated to different temperatures, we have conducted compositional analyses of raft and RDPM from rainbow trout Oncorhynchus mykiss Walbaum acclimated to 5°C and 20°C. We measured the composition of the major phospholipid classes and found differences in the rafts from the warm acclimated group compared with all other membrane fractions studied. Furthermore, we measured the fatty acid composition of the total lipid extracts as well as phosphatidylcholine (PtdCho) and phosphatidylethanolamine (PtdEtn) resolved from the membrane fractions. Acclimatory changes in unsaturation were larger in raft than RDPM and the patterns of change differed between PtdCho and PtdEtn.
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Materials and methods |
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Animals
Rainbow trout Oncorhynchus mykiss Walbaum were obtained from the
Alchesay National Fish Hatchery in Whiteriver, Arizona, and were maintained at
the Animal Resource Center of Arizona State University. Fish were housed in
recirculating freshwater aquaculture systems consisting of circular fiberglass
tanks; water temperatures were controlled using flow-through chillers. Animals
were acclimated to 5°C or 20°C for at least 3 weeks before use in
experiments. Fish were held under a constant 12 h:12 h L:D cycle and were fed
daily with Rangen Inc. (Buhl, ID, USA) trout food to satiation.
Plasma and raft membrane isolations
PMs were isolated from approximately 8 g of liver (pooled from several
fish) according to a modification of the procedure of Armstrong and Newman
(1985) as described previously
(Hazel et al., 1992
). The PM
was resuspended in working buffer (WB: 0.25 mol l-1 sucrose, 20
mmol l-1 tricine, pH 7.8, 1 mmol l-1 EDTA disodium salt)
and was separated into raft-depleted PM (RDPM) and raft-enriched PM (raft)
using a non-detergent based method (Smart
et al., 1995
). The lower buoyant density of raft, compared to
RDPM, was used as a means of separating the membrane fractions. Briefly, the
PM was sonicated and then brought to 23% (v/v) OptiPrep before layering a
10-20% (v/v) linear OptiPrep gradient on top (for a total of 11 ml in each
tube). After centrifuging for 90 min at 72 800 g in a Beckman
SW 41-Ti rotor (OP1) the top 5.5 ml (raft) were removed, mixed with 4 ml of
50% (v/v) OptiPrep in a fresh tube, overlaid with 250 µl 5% (v/v) OptiPrep,
and centrifuged for 90 min at 72 800 g (OP2). The raft
membrane concentrated at the top of the OP2 tube was collected with a Pasteur
pipette, diluted with three volumes of buffered saline and was centrifuged to
a pellet at 20 800 g for 20 min in a refrigerated
microcentrifuge (Eppendorf 5417 R). The bottom 5.5 ml from OP1 was diluted
with four volumes of buffered saline and was centrifuged for 1 h at 23 700
g in a Beckman JA 30.50 rotor to form the RDPM pellet.
Lipid extractions
Lipids were extracted from membrane samples using the method of Bligh and
Dyer (1959). Briefly, 2.4 ml of
aqueous membrane suspension was mixed with 9 ml of chloroform-methanol (1:2)
and incubated for 10 min. The addition of 3 ml of chloroform and 3 ml of 0.88%
aqueous KCl resulted in the formation of two phases. The lower hydrophobic
phase (containing the lipids) was recovered and transferred to a 50 ml boiling
flask. The solvent was driven off using a rotary evaporator. The sample was
dehydrated by adding 5 ml of absolute ethanol followed by rotary evaporation.
This procedure was repeated twice. The dried lipids were then transferred to
an autosampler vial using a small volume of chloroform-methanol (2:1).
Butylated hydroxytoluene (BHT) was added to a final concentration of 50 mg
l-1 to inhibit oxidation and the lipids were stored at
-20°C.
Protein and lipid analyses
Total protein was determined colorimetrically by the method of Bradford
(Bradford, 1976). Phospholipid
concentration was measured by colorimetric phosphate analysis after complete
hydrolysis of total lipid extracts or isolated PtdCho and PtdEtn
(Ames, 1966
). A coupled
cholesterol oxidase fluorometric assay was used to measure cholesterol
(Crockett and Hazel, 1995
).
Thin layer chromatography
Lipids were separated into major phospholipid classes using thin layer
chromatography (TLC). Lipids in chloroform-methanol (2:1) were spotted onto
the preabsorption zone of TLC plates (approximately 1.5 mg lipid per plate).
The solvent was driven off under nitrogen. The developing solvent was prepared
immediately prior to use: chloroform-methanol-acetic acid-water
(50:37.5:3:1.7; Holub and Skeaff,
1987). The developing solvent was poured into the TLC tank, fully
wetting the filter paper lining the sides of the tank. The dried TLC plates
were immediately placed in the tank, the top sealed, and developed for 1 h and
15 min.
After removal of the plates, residual solvent was dried under a stream of nitrogen. Each TLC plate was positioned between two plates of glass, leaving one edge of the TLC plate exposed. The exposed lipids were stained in an iodine chamber and identified against authentic standards. The separated lipid classes were recovered by scraping the silica gel (from the region of the plate not exposed to iodine) off the aluminum backing into test tubes. The lipid classes were extracted from the silica gel by adding 7 ml of chloroform-methanol (2:1), centrifuging briefly in a clinical centrifuge, and recovering the supernatant into a conical boiling flask. This procedure was repeated twice for a total volume of 21 ml solvent per sample, which was filtered through a 0.45 µm PTFE (polytetrafluoroethylene, commonly known as Teflon®) syringe filter. The solvent was driven off using a rotary evaporator and then a small volume of chloroform-methanol (2:1) was used to transfer the lipids to autosampler vials. BHT (50 mg l-1) was added and the vials were stored at -20°C.
Preparation of fatty acid methyl esters
Fatty acid methyl esters (FAMEs) were prepared from total lipid extracts
and TLC-separated lipid classes by the acid catalyzed method described by
Christie (2003). Lipids were
transferred from their storage vials to 15 ml glass tubes, the solvent was
driven off under nitrogen, and 2 ml of 3 M methanolic HCl added. The tubes
were placed in a heat block at 65°C, capped with a marble, and allowed to
reflux for 2 h. The tubes were removed and 5 ml of 5% (w/v) NaCl was added.
The FAMEs were extracted with 3.3 ml hexane three times, the hexane was pooled
in a fresh tube and then washed with 4 ml of 2% (w/v) KHCO3. The
hexane was then recovered to a fresh tube and dried with anhydrous sodium
sulfate. The hexane was again recovered to a fresh tube, leaving the sodium
sulfate behind, and the majority of the solvent driven off under nitrogen. The
remaining solvent was transferred to an autosampler vial, 50 mg l-1
BHT was added, and the vials were stored at -20°C.
Gas chromatography - Mass spectrometry
FAMEs were separated using a Varian (Palo Alto, CA, USA) Star 3400cx gas
chromatograph fitted with a Supelco SP 2380 column (30 mx0.25 mm
i.d.x0.2 µm film) with a helium carrier linear flow rate of 30 cm
s-1. The injector and detector manifold were at 250°C. The
column oven was maintained at 100°C for 2 min postinjection. The oven
temperature was then increased at a rate of 4°C min-1 for 30
min to a temperature of 220°C. At 30 min the oven temperature was
increased at 50°C min-1 to 250°C and held at this
temperature for 5 min to clear the column. The separated FAMEs were detected
using a Varian Saturn 4D mass spectrometer with a scan rate of 1000 ms with a
mass range between 50 and 400 m z-1. FAMEs were identified by
retention time and fragmentation pattern compared against authentic standards
and a fragmentation pattern library. Quantification was by peak integration
and expressed for individual FAMEs as percentages of the total in each
run.
Statistics
The percentages of each phospholipid class (e.g. phosphatidylcholine) from
raft and RDPM from both acclimation groups were analyzed using a one-way
analysis of variance (ANOVA) followed by Tukey's test for multiple
comparisons. For the fatty acids, the unsaturation index was calculated as the
number of double bonds per 100 fatty acids (the percentage abundance of each
fatty acid x number of double bonds, summed for all fatty acids).
Percentage unsaturation and unsaturation index were analyzed separately using
one-way ANOVAs and Tukey's test. In all cases a P value of 0.05 or
lower was accepted as indicating statistical significance.
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Results |
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Comparison of phospholipid classes in raft-enriched plasma membrane and raft-depleted plasma membrane
Raft and RDPM from cold-acclimated trout did not differ in phospholipid
class composition (Fig. 1C). In
both subdomains, PtdCho and PtdEtn were the major components while PtdSer,
PtdIns and SM each made up less than 10% of the total phospholipid. In
contrast, raft and RDPM did differ significantly in warm-acclimated fish: the
proportion of PtdSer was significantly higher in rafts (20%) than RDPM (6%;
P<0.001) whereas the proportion of PtdCho was lower in raft than
in RDPM (30% vs 46%; P=0.028;
Fig. 1D).
Fatty acid analyses
We next examined the fatty acid content of lipids extracted from raft and
RDPM from cold- and warm-acclimated fish. Fatty acid methyl esters (FAMEs)
were prepared from total lipid extracts and isolated PtdCho and PtdEtn, and
were analyzed with a gas chromatograph fitted with a mass spectrometer.
Seventeen major FAMEs were resolved. The major species resolved from the total
lipid extracts were, in order of abundance, 22:6n3 (26.8-29.2%), 16:0
(17.7-23.9%), 18:1n9 (13.1-15.3%), 18:0 (9.6-10.3%) and 18:2n6 (5.9-8.3%)
which, collectively, made up approximately 80% of the total FAMEs (see
complete data in Tables S1-S3 in supplementary material). We were primarily
interested in patterns of unsaturation since double bonds influence membrane
order and the interaction of phospholipids with cholesterol. We calculated two
indexes of fatty acid unsaturation. The unsaturation index (UI) is the number
of double bonds per 100 molecules of fatty acid. With this measure, either a
small number of highly unsaturated fatty acids or a large number of
monounsaturates can produce the same UI number. Therefore, we also calculated
the percentage of unsaturated fatty acids, i.e. the number of fatty acids with
one or more double bonds in a pool of 100. The data are presented on two
dimensional graphs with percentage unsaturation on the abscissa and the UI on
the ordinate. Additionally, because of their differing effects on membrane
physical properties, we analyzed the monounsaturated fatty acids (MUFA) and
polyunsaturated fatty acids (PUFA) separately, plotting the percentage of
fatty acids in each group in bar graphs.
The total fatty acid composition of raft and RDPM domains
We first examined the fatty acid composition of total lipid extracts of
raft and RDPM (Fig. 2). For
reference, in this and subsequent similar figures, isopleths are plotted
(dotted lines) representing the region on the graph where an increase in
percentage unsaturation is achieved by adding fatty acids with an average
specified number of double bonds (Logue et
al., 2000). For example, if all unsaturated fatty acids had three
double bonds, the lower isopleth in Fig.
2A would mark the UI for any given level of percentage
unsaturation. Increases in unsaturation that parallel an isopleth represent an
increase in the proportion of unsaturated fatty acids having the same average
number of double bonds, while those that cross isopleths represent a change in
the average number of double bonds per fatty acid.
|
Comparing total fatty acid compositions, there were no significant differences in UI among the samples (ANOVA P=0.411). However, there were differences in the percentage of unsaturated fatty acids (ANOVA P=0.008) with increased unsaturation in the samples from the cold-acclimated fish (Fig. 2A). This trend was small and not significant between the RDPMs (P=0.617), but the rafts did differ significantly (P=0.006), reflecting a greater acclimatory response. Raft and RDPM did not differ in percentage unsaturation in either cold-(P=0.399) or warm-(P=0.562) acclimated fish.
There were no significant differences in percentage MUFA among the samples (ANOVA P=0.082) (Fig. 2B). PUFA levels were slightly, but significantly lower (ANOVA P=0.024) in raft from warm-acclimated trout (44.1%) than raft (48.2%; P=0.045) or RDPM (48.6%; P=0.029) from cold-acclimated trout (Fig. 2C).
Phosphatidylcholine
We next examined changes in fatty acid composition of PtdCho during thermal
acclimation. There was a significant ANOVA (P=0.050) for percentage
unsaturated fatty acids, but there were no significant pairwise comparisons
among the samples (Fig. 3A).
The ANOVA of the UI data was significant (P=0.017) and the general
trend of the data reflected decreases in UI (across the isopleths;
N=4) with warm acclimation rather than changes in percentage
unsaturation. This pattern indicates that in PtdCho, acclimatory changes in
unsaturation occur primarily in the form of an increased number of double
bonds per unsaturated fatty acid, without change in the proportion of
unsaturated fatty acids. However, multiple comparisons demonstrated no
significant differences between the rafts (P=0.083) or the RDPMs
(P=0.141) and raft and RDPM had nearly identical UI values within
each acclimation group.
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Phosphatidylethanolamine
In PtdEtn, the trend with cold acclimation was for both UI and percentage
unsaturation to increase in parallel with the isopleths
(Fig. 4A), corresponding to an
increase in the proportion of unsaturated fatty acids, without a change in the
average number of double bonds. Although percentage unsaturation tended to
increase with cold acclimation in both raft and RDPM (ANOVA P=0.014),
this difference was only significant in rafts (P=0.036), not RDPMs
(P=0.161). Similarly, increases in UI with cold acclimation (ANOVA
P=0.037) were nearly significant in raft (P=0.061), but not
RDPM (P=0.274). There were no significant differences in the content
of MUFA (ANOVA P=0.896; Fig.
4B). The ANOVA for the PUFA was significant (P=0.039),
although there were no significant pair-wise comparisons
(Fig. 4C). However, the most
probable cause of the significant ANOVA was the 54.8-44.1% and 50.9-44.4%
decreases in PUFA with warm acclimation in raft and RDPM, respectively. This
is consistent with the modulation of the proportion of fatty acids carrying
between three and four double bonds, without altering the average number of
double bonds on the unsaturated fatty acids
(Fig. 4A).
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Discussion |
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However, the differences in fatty acid composition found in this study
(with thermal acclimation and between raft and RPDM) were surprisingly small.
This is difficult to reconcile with the conservation of physical properties
described in our previous studies and the theory that rafts are phase
separated from RDPM (Ahmed et al.,
1997). It is possible that there were larger changes in the fatty
acid content of sphingomyelin, which was not measured separately. It is also
important to point out that highly polar glycosphingolipids, which are
enriched in rafts (Fridriksson et al.,
1999
; Prinetti et al.,
2000
; Pike et al.,
2002
), would not have been efficiently recovered in our
preparation. Therefore, the contribution of this important class of lipids to
the raft fatty acid composition is unknown.
The second consistent pattern observed in this and in previous work was
greater differences between raft and RDPM from warm-acclimated than from
cold-acclimated trout. In the present study, this was clearly demonstrated by
the class compositional data. While there were no differences in the
cold-acclimated group, in the warm-acclimated group PtdSer was enriched in
raft compared to RDPM at the expense of PtdCho
(Fig. 1C,D). Cholesterol was
also more enriched in rafts of warm-acclimated than cold-acclimated fish
(Zehmer and Hazel, 2003).
Finally, in warm-acclimated trout, raft was more ordered than RDPM while in
the cold group, there was no difference in raft and RDPM order
(Zehmer and Hazel, 2004
).
A predominant model of lateral segregation of raft and RDPM is based on
liquid-ordered (Lo)/liquid-disordered (Ld) phase
separation (Ahmed et al.,
1997). This model predicts substantial differences in fatty acid
composition and order that were not observed. Therefore, it is puzzling how
raft and RDPM of cold-acclimated fish are segregated. However, it is important
to point out that the protein composition and membrane packing strength of
raft and RDPM are clearly distinct in both acclimation groups, as shown
previously (Zehmer and Hazel,
2003
). Therefore, raft and RPDM are distinct in properties and
some aspects of their composition, but it is not clear what forms the basis
for raft/RDPM segregation in PM of cold-acclimated trout.
A final pattern of interest in the current data involves the markedly
different patterns of change in acyl chain unsaturation between the major
phospholipids, PtdCho and PtdEtn. With cold acclimation, PtdEtn accumulated a
higher percentage of unsaturated fatty acids without a change in the average
number of double bonds (Fig. 4)
whereas in PtdCho more double bonds were introduced into the already
unsaturated fatty acids (Fig.
3). These results recapitulate those of Logue et al.
(2000), who undertook a
comparative study of brain synaptosomes isolated from animals adapted to
temperatures between -1°C and 41°C. Although the observation of this
phenomenon in brain and liver and in both thermal acclimation of a single
species (in both raft and RPDM) and adaptation across species, suggests it may
be of fundamental importance, the significance of this pattern is not
clear.
In summary, acclimatory compositional differences were greater in raft than in RDPM. These included changes in phospholipid class composition and patterns of fatty acid unsaturation. Additionally, there were differences in phospholipid composition between raft and RPDM only in the warm acclimation group. Surprisingly, there were no significant differences in fatty acid unsaturation between raft and RDPM in either acclimation group. The patterns of acclimatory changes in acyl chain unsaturation differed between PtdCho and PtdEtn. Collectively these and previously reported data suggest that regulation of raft properties may be especially important during thermal acclimation.
List of abbreviations
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Acknowledgments |
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Footnotes |
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References |
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