Articles in PresS. Am J Physiol Regul Integr Comp Physiol (December 17, 2014). doi:10.1152/ajpregu.00559.2013
1
Products of lipid peroxidation, but not membrane susceptibility to oxidative damage, are
2
conserved in skeletal muscle following temperature acclimation
3 4 5 6
Jeffrey M. Grim+*, Molly C. Semones+, Donald E. Kuhn+, Tamas Kriska#, Agnes Keszler##, and Elizabeth L. Crockett+
7 8 9
10 11 12 13 14 15
* Current Address: JMG (permanent address) Department of Biology, The University of Tampa, Tampa, FL, 33606, [email protected], Phone: 813-257-1807, Fax: 813-258-7496
17
##
+
Department of Biological Sciences, Ohio University, Athens, OH, 45701
16
#
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI, 53226
18
Running Head: Lipid peroxidation is not conserved following temperature acclimation
19 20
Running Head: LPO products are maintained with temperature acclimation
21 22
Keywords: oxidative stress, lipid peroxidation, phospholipid hydroperoxides, Vitamin E,
23
Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI, 53226
temperature acclimation
24 25
Copyright © 2014 by the American Physiological Society.
26
ABSTRACT
28
acclimation in ectothermic animals. Both responses may alter redox status and membrane
30
remodeling is sufficient to offset temperature-driven rates of LPO, and thus membrane
32
maintained over a range of physiological temperatures. To assess LPO susceptibility,
34
and sarcoplasmic reticulum from oxidative and glycolytic muscle of striped bass (Morone
36
contents of LPO products (i.e., individual classes of phospholipid hydroperoxides –
38
chain remodeling, these alterations do not counter the effect of temperature on LPO rates
40
phospholipid content and compared at a common temperature). Although muscles from
42
this difference is lost when PLOOH levels are normalized to total phospholipid. Contents
44
mitochondria prepared from oxidative muscle of warm-acclimated fish than those from
46
remodeling does not provide a means for offsetting thermal effects on rates of LPO,
48
temperature variation.
27
Changes in oxidative capacities and phospholipid remodeling accompany temperature
29
susceptibility to lipid peroxidation (LPO). We tested the hypothesis that phospholipid
31
susceptibility to LPO is conserved. We also predicted that the content of LPO products is
33
rates of LPO were quantified with the fluorescent probe C11-BODIPY in mitochondria
35
saxatilis) acclimated to 7° and 25°C. We also measured phospholipid compositions,
37
PLOOH), and two membrane antioxidants. Despite phospholipid head group and acyl
39
(i.e., LPO rates are generally not different among acclimation groups when normalized to
41
cold-acclimated fish have higher absolute levels of PLOOH than warm-acclimated fish,
43
of vitamin E and two homologs of ubiquinone are more than 4-times higher in
45
cold-acclimated animals. Collectively, our data demonstrate that although phospholipid
47
differences in phospholipid quantity ensure a constant proportion of LPO products with
49
INTRODUCTION
51
that may be inflicted upon biological molecules. Unless chain-breaking antioxidants (e.g.,
53
peroxidase) terminate the process, the reactions of LPO can self-propagate within
55
32, 44). LPO can disrupt the structure of biological membranes because LPO involves
57
positioning a polar element within the otherwise hydrophobic core of the membrane.
59
protein interactions, ultimately placing membrane integrity and function at risk (23, 26,
50
Lipid peroxidation (LPO) is unique among the various types of oxidative damage
52
vitamin E) and/or antioxidant enzymes (e.g., phospholipid hydroperoxide glutathione
54
biological membranes when oxidized lipids damage other membrane lipids (10, 21, 31,
56
the addition of polar hydroperoxy groups to both phospholipids and cholesterol,
58
These LPO-induced changes in biological membranes may alter lipid-lipid and lipid-
60
31).
62
temperatures, and vary widely for many temperate species (16). Ectotherms maintain
64
biological membranes (15) and altering capacities for oxidative metabolism (9).
66
phospholipids (12) and also possess increased ratios of phosphatidylethanolamine
68
in lipid composition confer some degree of constancy in the physical properties of
70
acclimated/adapted ectotherms at a greater risk of LPO-induced damage than the
72
susceptibility of LPO associated with lipid remodeling in cold-acclimated/adapted fishes
61
Body temperatures of ectothermic animals are determined by environmental
63
cellular function during temperature acclimation/adaptation in part by remodeling
65
Biological membranes from cold-acclimated/adapted fishes contain highly unsaturated
67
(PE)/phosphatidylcholine (PC) relative to warm-counterparts (13). While these changes
69
membranes, both of these compositional modifications may place membranes from cold-
71
membranes from warm-acclimated/adapted animals (3, 17, 46). An increased
73
may be further exacerbated by a heightened potential for generating reactive oxygen
75
(6, 22, 33) and increased oxidative capacities (9) compared with animals at warm body
77
While phospholipid remodeling and metabolic changes may increase the overall
74
species (ROS) because cold-bodied fishes often possess elevated mitochondrial densities
76
temperatures.
78
apparent risk of LPO at cold body temperatures (3, 17, 46), it is also possible that these
80
ROS production with temperature variation (4). Our major objective was to evaluate
82
rates of LPO in a manner that partially or completely offsets the effects of temperature.
84
focus on the response of the skeletal muscles to temperature acclimation because these
86
skeletal muscle has a more limited source of ROS compared to other tissues (e.g., liver),
88
redox system.
90
and sarcoplasmic reticulum were prepared from red (oxidative) and white (glycolytic)
92
system while monitoring the extent of LPO with the fluorometric probe C11-BODIPY in
94
tissue contents of phospholipid hydroperoxide (PLOOH) were quantified and also
79
alterations are beneficial, ensuring a steady rate (and/or amount) of LPO products and
81
whether the physiological changes associated with lipid remodeling are sufficient to alter
83
To do so, we utilized an ectothermic model, the striped bass (Morone saxatilis), with a
85
(this) tissues (organism) have (has) been previously characterized (5, 6, 36). Furthermore,
87
making it a more defined system to assess both pro- and antioxidant components of the
89
Striped bass were acclimated to 7° or 25°C, and membranes from mitochondria
91
skeletal muscle. Rates of LPO were quantified in vitro using a free radical-generating
93
both sets of membranes at common and physiological (acclimation) temperatures. Total
95
analyzed as discrete phospholipid classes using high-performance thin layer
96
chromatography (HPTLC) in order to assess the impact of acclimation temperature on
98
and acyl chain composition) and contents of neutral lipids involved in antioxidant defense
97
endogenous LPO products. Phospholipid compositions (analyzed by phospholipid class
99
(vitamin E and homologs of ubiquinone) were also characterized. This study utilizes a
101
neutral lipid remodeling with temperature acclimation affect rates of LPO and levels of
103
temperature acclimation contributes to preservation of both membrane susceptibility to
105
MATERIALS AND METHODS
100
comprehensive approach to examine how, and to what extent, both phospholipid and
102
LPO products. This study tests the hypothesis that membrane restructuring during
104
LPO and levels of LPO products.
106
Fish maintenance and acclimation
108
(Smyrna, Delaware) and housed at the Laboratory Animal Resources facility on the
110
equipped with biological, chemical, and UV filtration. Water quality parameters were
112
satiation with Zeigler’s Finfish Gold floating pellets throughout the periods prior to and
107
Juvenile striped bass, Morone saxatilis, were purchased from Delmarva Aquatics
109
campus of Ohio University in two 1200-L recirculating brackish water tanks (~5 ppt)
111
monitored daily and maintained with weekly partial water changes. Fish were fed daily to
113
during acclimations.
115
months before being used in the acclimation experiments. Following the initial growth
117
17°C. This intermediate temperature set point (intermediate to final acclimation
119
common thermal history of all experimental animals. After two weeks, the temperature of
114
Fish were held under ambient conditions (20°C ± 1°C, 12:12 L:D cycle) for 12
116
period, the temperature of each tank was decreased by 1°C/day to a final temperature of
118
temperatures of 7 and 25°C) was maintained for a period of two weeks to ensure a
120
each tank was changed by ±1°C/day until the final acclimation temperatures (7 or 25°C)
122
exposed to the final acclimation temperatures for 24 hours and showed no obvious signs
124
protocols used in this study (IACUC #13-L-011).
121
were reached. A six-week acclimation period commenced once animals had been
123
of stress. Institutional Animal Care and Use Committee at Ohio University approved all
125
Tissue Sampling and Membrane Preparation
127
respectively) were stunned with a cranial blow and euthanized by cervical transection.
129
(oxidative) and white (glycolytic) muscles were separated by dissection. Between 1.5-5 g
131
4-5 individuals and homogenized 10% (w/v) in a media containing 140 mM KCl, 20 mM
133
Biospec tissuemizer (three 5-sec bursts at low speed), and followed by 5 passes of a
135
enzymatic marker analyses (see below), and the remaining volume was used to prepare
137
respectively, described elsewhere (7). Final membrane preparations were divided into 6
139
All dissection, homogenization, and centrifugation steps were performed at 4°C to
126
Fish (mean mass = 19 ± 0.7 and 28 ± 1 g for 7°C and 25°C acclimated animals,
128
The entire axial muscle mass was removed from each side of the animal and red
130
(wet weight) each of oxidative and glycolytic muscles were pooled from approximately
132
Hepes, 10 mM EDTA, 0.1 mM EGTA, 5 mM MgCl2, 0.5% BSA (pH 7.1) first with a
134
Potter-Elvehjem homogenizer. A 1 mL aliquot of crude homogenate was saved for
136
mitochondrial membranes and SR following the modifications of (29, 30, 45),
138
aliquots that were snap-frozen in liquid nitrogen and stored at -70°C for further analysis.
140
minimize sample degradation.
142
(11, 39) and sarcoplasmic reticular Ca2+-ATP-ase (SERCA) for SR (40) as modified by
141
Activities of cytochrome c oxidase (CCO) for mitochondria (47) as modified by
143
(36) were measured to calculate enrichment factors to ensure comparable purities of
145
by an average of 6.3- and 8.3-times, respectively, between acclimation groups. All
147
spectrophotometer equipped with a circulating water bath.
144
membrane preparations between acclimation groups. Mitochondria and SR were enriched
146
enzymatic assays were conducted at 20°C using a Beckman 640 UV/VIS
148
Susceptibility to lipid peroxidation (LPO) - BODIPY
150
eight individual membrane preparations per temperature treatment by following the rate
152
1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid) as described
154
concentration of 148 nM with a 0.05 mg/ml membrane solution, and the probe/membrane
156
both mitochondria and SR using hydroxyl radicals produced by the Fenton reaction
158
final concentrations of 13 and 52 µM, respectively. Probe oxidation was monitored for all
160
respectively and linear portions of the decay slope were recorded as the rate of LPO (Δ
162
all assay temperatures, and therefore was subtracted from rates in the presence of LPO
164
phospholipid and protein content by measuring hydrolyzable phosphate (37) and total
149
The susceptibility of biological membranes to lipid peroxidation was quantified in
151
of oxidation of the fluorescent probe C11-BODIPY® 581/581 (4,4,-difluoro-5-(4-phenyl-
153
previously (7). Briefly, a C11-BODIPY solution (10 µM) was diluted to a final
155
mixture was stirred for 60 min in the dark at 4°C. Subsequently, LPO was induced in
157
between Cu2+ (as CuSO4) and cumene hydroperoxide (CumOOH) in a 1:4 ratio (14) to
159
membranes at 7, 16, and 25°C with excitation/emission wavelengths of 568/590 nm,
161
fluorescence intensity Δ min-1). Autoxidation was detectable for both membrane types at
163
induction system in all final calculations. All rates of LPO were normalized to
165
protein (41).
166
Phospholipid compositional analysis
168
of the same eight preparations of intracellular membranes used for the quantification of
170
at the Kansas State University Lipidomics Research Center in order to quantify the
172
Membrane unsaturation index (UI) was calculated following the modification of Hulbert
174
in diacyl phospholipids, relative to the 6 in individual fatty acyl chains.
167
Following the methods of Bligh and Dyer (1), lipids were extracted from aliquots
169
LPO susceptibility. Lipid extracts were analyzed by triple-quadrupole mass spectrometry
171
relative abundance of phospholipid headgroups and molecular species of phospholipids.
173
et al (20), described in Grim et al (7), to account for the potential 12 double bonds present
175
Quantification of tocopherol (Vitamin E) and ubiquinones (CoQ)
177
(BHT) was added to each of ten mitochondrial membrane samples from red muscle (2µl
179
vol/vol), and lipid soluble compounds were extracted with n-hexane (2:1, vol/vol). After
181
evaporated under nitrogen. Dried samples were immediately dissolved in 100µl ethanol,
183
0.5µm particle size) HPLC column. Tocopherol and ubiquinone were eluted with
185
Quantification was performed based on detector response of known concentrations and
187
7.8 min, and CoQ9 – 16 min) of authentic standard compounds. Tocopherol and
176
To prevent the oxidation of tocopherol and ubiquinone, butylated hydroxytoluene
178
of 1mM BHT in ethanol/100µl). Samples were deprotonized with 100% ethanol (1:1,
180
centrifugation at 5min/8000gav/4°C, the organic supernatant was collected and the hexane
182
and 50µl of sample were injected onto an unmodified Kromasil C18 (25cm x 0.46cm,
184
hexane:methanol (15:85) at a flow rate of 1 ml/min and detected at 275nm.
186
retention times (tocopherol – 5.4 min, an unidentified homolog of ubiquinone, CoQ? –
188
ubiquinone amounts were normalized to phospholipid content.
189
Quantification of endogenous phospholipid hydroperoxides (PLOOH)
191
above for dissection methods) using modifications of Bligh and Dyer (1). If not used
193
glass vials.
195
presence of five volumes of ice-cold acetone under a nitrogen atmosphere at -20°C for 30
197
20°C, and the resulting precipitate was collected. Both precipitates were dried completely
199
LPO) or hexane with 3% isopropyl alcohol (IPA) (for HPTLC analyses to compare
201
stored at -20°C under a nitrogen atmosphere in Teflon capped glass vials. This extraction
203
total lipid extracts.
205
classes was modified from Kriska and Girotti (25). Dried phospholipid samples were re-
207
under high pressure onto glass Silica G chromatography plates (Si60; EMD) using a
209
Separation was carried out with a mobile phase solvent consisting of chloroform,
211
developed plates were removed from the chamber and dried under argon gas. Dried plates
190 192 194
Lipids were extracted from previously collected axial red and white muscle (see
immediately, samples were stored at -20°C under a nitrogen atmosphere in Teflon capped
Phospholipids were precipitated from total lipids (dissolved in hexane) in the
196
minutes. The decanted acetone was allowed to stand for an additional 30 minutes at -
198
under nitrogen gas, re-suspended in either chloroform (CHCl3) (for quantification of total
200
PLOOH among PL classes), and finally combined. Samples not immediately used were
202
protocol resulted in a high efficacy of phospholipid recovery (between 95-99%) from
204
Partitioning of phospholipid hydroperoxides (PLOOH) among phospholipid
206
suspended in 50-200 µl of hexane:isopropyl alcohol (3% IPA), and samples were spotted
208
Linomat5 semiautomatic sample applicator (Camag Scientific, Wilmington, N.C.).
210
methanol, acetic acid, and water (CHCl3:MeOH:HOAc:H2O as 100:75:7:4), and
212
were then sprayed with a N,N,N1,N1-tetramethyl-p-phenylenediamine solution (250 mg
214
were visible. Bands continued to develop under argon gas until their maximum intensity
216
band intensities were quantified using Gel Analyzer (freeware by Dr. Istvan Lazar)
218
HpODE; Cayman Chemicals, Ann Arbor, MI).
220
isolates were measured using a commercial kit (Cayman Chemical, Ann Arbor, MI). The
222
described above. The absorbance of assay reactions was measured at 500 nm with a
224
curve of (13-HpODE).
213
TPD in 12.5 mls each of MeOH and H2O, and 0.25 ml HOAc) until faint purple bands
215
was reached. Plates were photographed on a covered light box using a 12 MP camera and
217
against a standard curve of the PLOOH 13-hydroperoxy-9,11E-octadecadienoic (13-
219
Total phospholipid hydroperoxide (PLOOH - LPO) amounts from phospholipid
221
manufacturer’s protocol was followed with the exception of sample preparation as
223
Beckman DU640 spectrophotometer. All samples were compared to a PLOOH standard
225
Statistical Analyses
227
PLOOH, levels of tocopherol (Vitamin E) and ubiquinone, and all phospholipid data
229
t-tests or with Mann-Whitney tests when assumptions of normality and/or homogeneity
231
Bartlett’s test, respectively) (GraphPad Prism version 6.0, GraphPad Software, La Jolla,
233
noted, data are presented as means ± standard error of the mean (s.e.m.).
226
LPO susceptibility (as slope of fluorescence decay), total partitioned levels of
228
were compared between temperature acclimation groups using either parametric unpaired
230
of variance were violated (as determined by D’Agostino-Pearson omnibus test and
232
CA). All statistical conclusions were based on alpha values (α) of 0.05. Unless otherwise
234
235
RESULTS
236
Susceptibility to LPO
238
phospholipid content are generally similar between acclimation groups, regardless of
240
prepared from white muscle, in which rates of LPO in membranes from warm-acclimated
242
assay temperatures compared with mitochondrial membranes from cold-acclimated fish
237
When compared at common temperatures, rates of membrane LPO normalized to
239
assay temperature or membrane type (Fig 1). An exception are mitochondrial membranes
241
individuals are higher, by an average of 1.8-times, at low (7°C) and intermediate (16°C)
243
(Fig. 1B; t-test; p < 0.05).
245
prepared from both red (oxidative) and white (glycolytic) muscle are affected by
247
acclimated fish are on average higher by 1.4- and 2.1-times in mitochondria and
249
relative to membranes from warm-acclimated fish (Figs. 2A,C; t-test; p < 0.05). In
251
acclimated animals are higher by an average of 1.8- and 1.9-times for mitochondria and
253
cold-acclimated individuals (Figs. 2B,D; t-test; p < 0.05).
244
When normalized to membrane protein, rates of LPO in membrane fractions
246
acclimation temperature (Fig. 2). Rates of LPO in membranes from red muscle of cold-
248
sarcoplasmic reticulum (SR), respectively, at all assay temperatures (7°C, 16°C, 25°C)
250
contrast, rates of LPO in membrane fractions prepared from white muscle in warm-
252
SR at all assay temperatures, respectively, compared with membranes prepared from
254
Quantification of endogenous PLOOH
256
weight, is 1.7-times higher in both red and white muscle from animals at an acclimation
255 257
The total content of phospholipid hydroperoxide (PLOOH), normalized to tissue
temperature of 7°C compared to those at 25°C (Fig. 3A; t-test; p < 0.05). However, when
258
the content of PLOOH is normalized to content of phospholipid, there are no significant
260
25°C; Fig. 3B) or muscle types (red versus white; data not shown). The higher absolute
262
animals are driven in large part by the increase (2.5-times) in PLOOH containing the
264
rise (1.7-times) in PLOOH containing the choline headgroup (i.e., PCOOH) (Fig. 4A; t-
259
differences for comparisons made either between acclimation temperatures (7°C versus
261
levels of PLOOH (i.e., on a per gram basis) for muscular tissues of cold-acclimated
263
ethanolamine headgroup (i.e., PEOOH) (Fig. 4A; Mann-Whitney test; p < 0.05) and the
265
test; p < 0.05) for red and white muscle, respectively.
267
phosphatidylethanolamine – PE, phosphatidylcholine – PC, phosphatidylinositol – PI,
269
significant differences among temperature treatments for individual PLOOH classes
271
in oxidation of cardiolipin (CL) of red muscle of warm-acclimated fish compared with
266
PLOOHs partition among five major phospholipid classes (cardiolipin – CL,
268
and phosphatidylserine – PS). Similar to the trend for total PLOOH, there are no
270
relative to total phospholipid content. One exception, however, is the 1.5-times increase
272
cold-acclimated fish (Fig. 4B).
274
a muscle type and also between red and white muscles. The rank order of oxidized
276
> PI > PS > CL. In contrast, the ranking of PLOOH for red muscle varies between
278
25°C). These results demonstrate an increased oxidation of CL in red muscle compared
280
oxidative (red) fibers.
273
The rank order of PLOOH by class is different among acclimation groups within
275
phospholipids for white muscle from both 7°C and 25°C acclimated animals is PE > PC
277
acclimation groups (PE > PC > CL > PI > PS for 7°C and PC > PE > CL > PI > PS for
279
with white muscle, which is likely explained by higher contents of mitochondria in
281
Levels of Vitamin E and Ubiquinone
283
altered by temperature acclimation. Vitamin E, CoQ9, and an unidentified ubiquinone
285
mitochondrial membranes of warm-acclimated animals, when compared to cold-
287
Phospholipid Composition
289
groups in membranes from both red and white muscle (Tables 1 and 2). Although
291
other phospholipid classes are identifiable in both membranes, and the majority of these
293
from cold-acclimated fish are enriched in PE, relative to PC. Mitochondrial membranes
295
higher relative to warm-acclimated individuals, while SR membranes from the cold-
297
6A and 6D; t-test; p < 0.05).
299
altered by temperature acclimation in both mitochondria and SR (Tables 3 and 4). Higher
301
raise the unsaturation index (UI) of some phospholipid classes, including PC (SR only)
303
acclimated animals than cold-acclimated fish (Tables 5 and 6). More specifically,
282
Levels of vitamin E and ubiquinone (CoQ) in mitochondrial membranes are
284
homologue (CoQ?) relative to phospholipid contents are 5- to 3-times higher in
286
acclimated counterparts (Fig. 5; t-test; p < 0.05).
288
Temperature acclimation induces significant remodeling of phospholipid head
290
mitochondria and SR are dominated by phospholipids with PE and PC headgroups, nine
292
vary in response to temperature acclimation (Tables 1 and 2). As expected, membranes
294
from cold-acclimated animals have ratios of PE-to-PC that are on average 1.8-times
296
acclimated group show a more modest 1.4-times increase, on average, in this ratio (Fig.
298
Phospholipids varying in contents of saturated and unsaturated fatty acids are also
300
amounts of polyunsaturated fatty acids (PUFA) following cold-acclimation significantly
302
and PE (both membranes), while the UI in PS is higher in both membranes from warm-
304
mitochondrial membranes from red muscle following cold-acclimation have a 1.4-times
306
mitochondria prepared from cold-acclimated animals (Fig. 6B; t-test; p < 0.05), while SR
308
0.0001). While the magnitude of response is less than in red muscle, total UI of both
310
increased by a modest 10%, relative to animals at warm body temperature (Fig. 6E and
312
DISCUSSION
314
and levels of acyl chain unsaturation during thermal acclimation is not sufficient to offset
316
membranes to LPO is not conserved with temperature variation. In fact, patterns of
318
of striped bass (Morone saxatilis). Even with elevated levels of unsaturated fatty acyl
320
LPO normalized to phospholipid content are generally similar (red muscle) or even lower
322
25°C animals. However, employing a different normalization criterion to rates of LPO
324
of LPO and levels of membrane protein do occur with temperature acclimation. These
326
(e.g.,(6) and Table 7 ), and indicate that the choice of normalization criteria can influence
305
higher UI of PE phospholipids, leading to a total increase in UI of 1.2-times in
307
has 1.2- and 1.3-times higher UI for PC and PE, respectively, (Fig. 6C; t-test; p
1
Products of lipid peroxidation, but not membrane susceptibility to oxidative damage, are
2
conserved in skeletal muscle following temperature acclimation
3 4 5 6
Jeffrey M. Grim+*, Molly C. Semones+, Donald E. Kuhn+, Tamas Kriska#, Agnes Keszler##, and Elizabeth L. Crockett+
7 8 9
10 11 12 13 14 15
* Current Address: JMG (permanent address) Department of Biology, The University of Tampa, Tampa, FL, 33606, [email protected], Phone: 813-257-1807, Fax: 813-258-7496
17
##
+
Department of Biological Sciences, Ohio University, Athens, OH, 45701
16
#
Department of Biochemistry, Medical College of Wisconsin, Milwaukee, WI, 53226
18
Running Head: Lipid peroxidation is not conserved following temperature acclimation
19 20
Running Head: LPO products are maintained with temperature acclimation
21 22
Keywords: oxidative stress, lipid peroxidation, phospholipid hydroperoxides, Vitamin E,
23
Department of Anesthesiology, Medical College of Wisconsin, Milwaukee, WI, 53226
temperature acclimation
24 25
Copyright © 2014 by the American Physiological Society.
26
ABSTRACT
28
acclimation in ectothermic animals. Both responses may alter redox status and membrane
30
remodeling is sufficient to offset temperature-driven rates of LPO, and thus membrane
32
maintained over a range of physiological temperatures. To assess LPO susceptibility,
34
and sarcoplasmic reticulum from oxidative and glycolytic muscle of striped bass (Morone
36
contents of LPO products (i.e., individual classes of phospholipid hydroperoxides –
38
chain remodeling, these alterations do not counter the effect of temperature on LPO rates
40
phospholipid content and compared at a common temperature). Although muscles from
42
this difference is lost when PLOOH levels are normalized to total phospholipid. Contents
44
mitochondria prepared from oxidative muscle of warm-acclimated fish than those from
46
remodeling does not provide a means for offsetting thermal effects on rates of LPO,
48
temperature variation.
27
Changes in oxidative capacities and phospholipid remodeling accompany temperature
29
susceptibility to lipid peroxidation (LPO). We tested the hypothesis that phospholipid
31
susceptibility to LPO is conserved. We also predicted that the content of LPO products is
33
rates of LPO were quantified with the fluorescent probe C11-BODIPY in mitochondria
35
saxatilis) acclimated to 7° and 25°C. We also measured phospholipid compositions,
37
PLOOH), and two membrane antioxidants. Despite phospholipid head group and acyl
39
(i.e., LPO rates are generally not different among acclimation groups when normalized to
41
cold-acclimated fish have higher absolute levels of PLOOH than warm-acclimated fish,
43
of vitamin E and two homologs of ubiquinone are more than 4-times higher in
45
cold-acclimated animals. Collectively, our data demonstrate that although phospholipid
47
differences in phospholipid quantity ensure a constant proportion of LPO products with
49
INTRODUCTION
51
that may be inflicted upon biological molecules. Unless chain-breaking antioxidants (e.g.,
53
peroxidase) terminate the process, the reactions of LPO can self-propagate within
55
32, 44). LPO can disrupt the structure of biological membranes because LPO involves
57
positioning a polar element within the otherwise hydrophobic core of the membrane.
59
protein interactions, ultimately placing membrane integrity and function at risk (23, 26,
50
Lipid peroxidation (LPO) is unique among the various types of oxidative damage
52
vitamin E) and/or antioxidant enzymes (e.g., phospholipid hydroperoxide glutathione
54
biological membranes when oxidized lipids damage other membrane lipids (10, 21, 31,
56
the addition of polar hydroperoxy groups to both phospholipids and cholesterol,
58
These LPO-induced changes in biological membranes may alter lipid-lipid and lipid-
60
31).
62
temperatures, and vary widely for many temperate species (16). Ectotherms maintain
64
biological membranes (15) and altering capacities for oxidative metabolism (9).
66
phospholipids (12) and also possess increased ratios of phosphatidylethanolamine
68
in lipid composition confer some degree of constancy in the physical properties of
70
acclimated/adapted ectotherms at a greater risk of LPO-induced damage than the
72
susceptibility of LPO associated with lipid remodeling in cold-acclimated/adapted fishes
61
Body temperatures of ectothermic animals are determined by environmental
63
cellular function during temperature acclimation/adaptation in part by remodeling
65
Biological membranes from cold-acclimated/adapted fishes contain highly unsaturated
67
(PE)/phosphatidylcholine (PC) relative to warm-counterparts (13). While these changes
69
membranes, both of these compositional modifications may place membranes from cold-
71
membranes from warm-acclimated/adapted animals (3, 17, 46). An increased
73
may be further exacerbated by a heightened potential for generating reactive oxygen
75
(6, 22, 33) and increased oxidative capacities (9) compared with animals at warm body
77
While phospholipid remodeling and metabolic changes may increase the overall
74
species (ROS) because cold-bodied fishes often possess elevated mitochondrial densities
76
temperatures.
78
apparent risk of LPO at cold body temperatures (3, 17, 46), it is also possible that these
80
ROS production with temperature variation (4). Our major objective was to evaluate
82
rates of LPO in a manner that partially or completely offsets the effects of temperature.
84
focus on the response of the skeletal muscles to temperature acclimation because these
86
skeletal muscle has a more limited source of ROS compared to other tissues (e.g., liver),
88
redox system.
90
and sarcoplasmic reticulum were prepared from red (oxidative) and white (glycolytic)
92
system while monitoring the extent of LPO with the fluorometric probe C11-BODIPY in
94
tissue contents of phospholipid hydroperoxide (PLOOH) were quantified and also
79
alterations are beneficial, ensuring a steady rate (and/or amount) of LPO products and
81
whether the physiological changes associated with lipid remodeling are sufficient to alter
83
To do so, we utilized an ectothermic model, the striped bass (Morone saxatilis), with a
85
(this) tissues (organism) have (has) been previously characterized (5, 6, 36). Furthermore,
87
making it a more defined system to assess both pro- and antioxidant components of the
89
Striped bass were acclimated to 7° or 25°C, and membranes from mitochondria
91
skeletal muscle. Rates of LPO were quantified in vitro using a free radical-generating
93
both sets of membranes at common and physiological (acclimation) temperatures. Total
95
analyzed as discrete phospholipid classes using high-performance thin layer
96
chromatography (HPTLC) in order to assess the impact of acclimation temperature on
98
and acyl chain composition) and contents of neutral lipids involved in antioxidant defense
97
endogenous LPO products. Phospholipid compositions (analyzed by phospholipid class
99
(vitamin E and homologs of ubiquinone) were also characterized. This study utilizes a
101
neutral lipid remodeling with temperature acclimation affect rates of LPO and levels of
103
temperature acclimation contributes to preservation of both membrane susceptibility to
105
MATERIALS AND METHODS
100
comprehensive approach to examine how, and to what extent, both phospholipid and
102
LPO products. This study tests the hypothesis that membrane restructuring during
104
LPO and levels of LPO products.
106
Fish maintenance and acclimation
108
(Smyrna, Delaware) and housed at the Laboratory Animal Resources facility on the
110
equipped with biological, chemical, and UV filtration. Water quality parameters were
112
satiation with Zeigler’s Finfish Gold floating pellets throughout the periods prior to and
107
Juvenile striped bass, Morone saxatilis, were purchased from Delmarva Aquatics
109
campus of Ohio University in two 1200-L recirculating brackish water tanks (~5 ppt)
111
monitored daily and maintained with weekly partial water changes. Fish were fed daily to
113
during acclimations.
115
months before being used in the acclimation experiments. Following the initial growth
117
17°C. This intermediate temperature set point (intermediate to final acclimation
119
common thermal history of all experimental animals. After two weeks, the temperature of
114
Fish were held under ambient conditions (20°C ± 1°C, 12:12 L:D cycle) for 12
116
period, the temperature of each tank was decreased by 1°C/day to a final temperature of
118
temperatures of 7 and 25°C) was maintained for a period of two weeks to ensure a
120
each tank was changed by ±1°C/day until the final acclimation temperatures (7 or 25°C)
122
exposed to the final acclimation temperatures for 24 hours and showed no obvious signs
124
protocols used in this study (IACUC #13-L-011).
121
were reached. A six-week acclimation period commenced once animals had been
123
of stress. Institutional Animal Care and Use Committee at Ohio University approved all
125
Tissue Sampling and Membrane Preparation
127
respectively) were stunned with a cranial blow and euthanized by cervical transection.
129
(oxidative) and white (glycolytic) muscles were separated by dissection. Between 1.5-5 g
131
4-5 individuals and homogenized 10% (w/v) in a media containing 140 mM KCl, 20 mM
133
Biospec tissuemizer (three 5-sec bursts at low speed), and followed by 5 passes of a
135
enzymatic marker analyses (see below), and the remaining volume was used to prepare
137
respectively, described elsewhere (7). Final membrane preparations were divided into 6
139
All dissection, homogenization, and centrifugation steps were performed at 4°C to
126
Fish (mean mass = 19 ± 0.7 and 28 ± 1 g for 7°C and 25°C acclimated animals,
128
The entire axial muscle mass was removed from each side of the animal and red
130
(wet weight) each of oxidative and glycolytic muscles were pooled from approximately
132
Hepes, 10 mM EDTA, 0.1 mM EGTA, 5 mM MgCl2, 0.5% BSA (pH 7.1) first with a
134
Potter-Elvehjem homogenizer. A 1 mL aliquot of crude homogenate was saved for
136
mitochondrial membranes and SR following the modifications of (29, 30, 45),
138
aliquots that were snap-frozen in liquid nitrogen and stored at -70°C for further analysis.
140
minimize sample degradation.
142
(11, 39) and sarcoplasmic reticular Ca2+-ATP-ase (SERCA) for SR (40) as modified by
141
Activities of cytochrome c oxidase (CCO) for mitochondria (47) as modified by
143
(36) were measured to calculate enrichment factors to ensure comparable purities of
145
by an average of 6.3- and 8.3-times, respectively, between acclimation groups. All
147
spectrophotometer equipped with a circulating water bath.
144
membrane preparations between acclimation groups. Mitochondria and SR were enriched
146
enzymatic assays were conducted at 20°C using a Beckman 640 UV/VIS
148
Susceptibility to lipid peroxidation (LPO) - BODIPY
150
eight individual membrane preparations per temperature treatment by following the rate
152
1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid) as described
154
concentration of 148 nM with a 0.05 mg/ml membrane solution, and the probe/membrane
156
both mitochondria and SR using hydroxyl radicals produced by the Fenton reaction
158
final concentrations of 13 and 52 µM, respectively. Probe oxidation was monitored for all
160
respectively and linear portions of the decay slope were recorded as the rate of LPO (Δ
162
all assay temperatures, and therefore was subtracted from rates in the presence of LPO
164
phospholipid and protein content by measuring hydrolyzable phosphate (37) and total
149
The susceptibility of biological membranes to lipid peroxidation was quantified in
151
of oxidation of the fluorescent probe C11-BODIPY® 581/581 (4,4,-difluoro-5-(4-phenyl-
153
previously (7). Briefly, a C11-BODIPY solution (10 µM) was diluted to a final
155
mixture was stirred for 60 min in the dark at 4°C. Subsequently, LPO was induced in
157
between Cu2+ (as CuSO4) and cumene hydroperoxide (CumOOH) in a 1:4 ratio (14) to
159
membranes at 7, 16, and 25°C with excitation/emission wavelengths of 568/590 nm,
161
fluorescence intensity Δ min-1). Autoxidation was detectable for both membrane types at
163
induction system in all final calculations. All rates of LPO were normalized to
165
protein (41).
166
Phospholipid compositional analysis
168
of the same eight preparations of intracellular membranes used for the quantification of
170
at the Kansas State University Lipidomics Research Center in order to quantify the
172
Membrane unsaturation index (UI) was calculated following the modification of Hulbert
174
in diacyl phospholipids, relative to the 6 in individual fatty acyl chains.
167
Following the methods of Bligh and Dyer (1), lipids were extracted from aliquots
169
LPO susceptibility. Lipid extracts were analyzed by triple-quadrupole mass spectrometry
171
relative abundance of phospholipid headgroups and molecular species of phospholipids.
173
et al (20), described in Grim et al (7), to account for the potential 12 double bonds present
175
Quantification of tocopherol (Vitamin E) and ubiquinones (CoQ)
177
(BHT) was added to each of ten mitochondrial membrane samples from red muscle (2µl
179
vol/vol), and lipid soluble compounds were extracted with n-hexane (2:1, vol/vol). After
181
evaporated under nitrogen. Dried samples were immediately dissolved in 100µl ethanol,
183
0.5µm particle size) HPLC column. Tocopherol and ubiquinone were eluted with
185
Quantification was performed based on detector response of known concentrations and
187
7.8 min, and CoQ9 – 16 min) of authentic standard compounds. Tocopherol and
176
To prevent the oxidation of tocopherol and ubiquinone, butylated hydroxytoluene
178
of 1mM BHT in ethanol/100µl). Samples were deprotonized with 100% ethanol (1:1,
180
centrifugation at 5min/8000gav/4°C, the organic supernatant was collected and the hexane
182
and 50µl of sample were injected onto an unmodified Kromasil C18 (25cm x 0.46cm,
184
hexane:methanol (15:85) at a flow rate of 1 ml/min and detected at 275nm.
186
retention times (tocopherol – 5.4 min, an unidentified homolog of ubiquinone, CoQ? –
188
ubiquinone amounts were normalized to phospholipid content.
189
Quantification of endogenous phospholipid hydroperoxides (PLOOH)
191
above for dissection methods) using modifications of Bligh and Dyer (1). If not used
193
glass vials.
195
presence of five volumes of ice-cold acetone under a nitrogen atmosphere at -20°C for 30
197
20°C, and the resulting precipitate was collected. Both precipitates were dried completely
199
LPO) or hexane with 3% isopropyl alcohol (IPA) (for HPTLC analyses to compare
201
stored at -20°C under a nitrogen atmosphere in Teflon capped glass vials. This extraction
203
total lipid extracts.
205
classes was modified from Kriska and Girotti (25). Dried phospholipid samples were re-
207
under high pressure onto glass Silica G chromatography plates (Si60; EMD) using a
209
Separation was carried out with a mobile phase solvent consisting of chloroform,
211
developed plates were removed from the chamber and dried under argon gas. Dried plates
190 192 194
Lipids were extracted from previously collected axial red and white muscle (see
immediately, samples were stored at -20°C under a nitrogen atmosphere in Teflon capped
Phospholipids were precipitated from total lipids (dissolved in hexane) in the
196
minutes. The decanted acetone was allowed to stand for an additional 30 minutes at -
198
under nitrogen gas, re-suspended in either chloroform (CHCl3) (for quantification of total
200
PLOOH among PL classes), and finally combined. Samples not immediately used were
202
protocol resulted in a high efficacy of phospholipid recovery (between 95-99%) from
204
Partitioning of phospholipid hydroperoxides (PLOOH) among phospholipid
206
suspended in 50-200 µl of hexane:isopropyl alcohol (3% IPA), and samples were spotted
208
Linomat5 semiautomatic sample applicator (Camag Scientific, Wilmington, N.C.).
210
methanol, acetic acid, and water (CHCl3:MeOH:HOAc:H2O as 100:75:7:4), and
212
were then sprayed with a N,N,N1,N1-tetramethyl-p-phenylenediamine solution (250 mg
214
were visible. Bands continued to develop under argon gas until their maximum intensity
216
band intensities were quantified using Gel Analyzer (freeware by Dr. Istvan Lazar)
218
HpODE; Cayman Chemicals, Ann Arbor, MI).
220
isolates were measured using a commercial kit (Cayman Chemical, Ann Arbor, MI). The
222
described above. The absorbance of assay reactions was measured at 500 nm with a
224
curve of (13-HpODE).
213
TPD in 12.5 mls each of MeOH and H2O, and 0.25 ml HOAc) until faint purple bands
215
was reached. Plates were photographed on a covered light box using a 12 MP camera and
217
against a standard curve of the PLOOH 13-hydroperoxy-9,11E-octadecadienoic (13-
219
Total phospholipid hydroperoxide (PLOOH - LPO) amounts from phospholipid
221
manufacturer’s protocol was followed with the exception of sample preparation as
223
Beckman DU640 spectrophotometer. All samples were compared to a PLOOH standard
225
Statistical Analyses
227
PLOOH, levels of tocopherol (Vitamin E) and ubiquinone, and all phospholipid data
229
t-tests or with Mann-Whitney tests when assumptions of normality and/or homogeneity
231
Bartlett’s test, respectively) (GraphPad Prism version 6.0, GraphPad Software, La Jolla,
233
noted, data are presented as means ± standard error of the mean (s.e.m.).
226
LPO susceptibility (as slope of fluorescence decay), total partitioned levels of
228
were compared between temperature acclimation groups using either parametric unpaired
230
of variance were violated (as determined by D’Agostino-Pearson omnibus test and
232
CA). All statistical conclusions were based on alpha values (α) of 0.05. Unless otherwise
234
235
RESULTS
236
Susceptibility to LPO
238
phospholipid content are generally similar between acclimation groups, regardless of
240
prepared from white muscle, in which rates of LPO in membranes from warm-acclimated
242
assay temperatures compared with mitochondrial membranes from cold-acclimated fish
237
When compared at common temperatures, rates of membrane LPO normalized to
239
assay temperature or membrane type (Fig 1). An exception are mitochondrial membranes
241
individuals are higher, by an average of 1.8-times, at low (7°C) and intermediate (16°C)
243
(Fig. 1B; t-test; p < 0.05).
245
prepared from both red (oxidative) and white (glycolytic) muscle are affected by
247
acclimated fish are on average higher by 1.4- and 2.1-times in mitochondria and
249
relative to membranes from warm-acclimated fish (Figs. 2A,C; t-test; p < 0.05). In
251
acclimated animals are higher by an average of 1.8- and 1.9-times for mitochondria and
253
cold-acclimated individuals (Figs. 2B,D; t-test; p < 0.05).
244
When normalized to membrane protein, rates of LPO in membrane fractions
246
acclimation temperature (Fig. 2). Rates of LPO in membranes from red muscle of cold-
248
sarcoplasmic reticulum (SR), respectively, at all assay temperatures (7°C, 16°C, 25°C)
250
contrast, rates of LPO in membrane fractions prepared from white muscle in warm-
252
SR at all assay temperatures, respectively, compared with membranes prepared from
254
Quantification of endogenous PLOOH
256
weight, is 1.7-times higher in both red and white muscle from animals at an acclimation
255 257
The total content of phospholipid hydroperoxide (PLOOH), normalized to tissue
temperature of 7°C compared to those at 25°C (Fig. 3A; t-test; p < 0.05). However, when
258
the content of PLOOH is normalized to content of phospholipid, there are no significant
260
25°C; Fig. 3B) or muscle types (red versus white; data not shown). The higher absolute
262
animals are driven in large part by the increase (2.5-times) in PLOOH containing the
264
rise (1.7-times) in PLOOH containing the choline headgroup (i.e., PCOOH) (Fig. 4A; t-
259
differences for comparisons made either between acclimation temperatures (7°C versus
261
levels of PLOOH (i.e., on a per gram basis) for muscular tissues of cold-acclimated
263
ethanolamine headgroup (i.e., PEOOH) (Fig. 4A; Mann-Whitney test; p < 0.05) and the
265
test; p < 0.05) for red and white muscle, respectively.
267
phosphatidylethanolamine – PE, phosphatidylcholine – PC, phosphatidylinositol – PI,
269
significant differences among temperature treatments for individual PLOOH classes
271
in oxidation of cardiolipin (CL) of red muscle of warm-acclimated fish compared with
266
PLOOHs partition among five major phospholipid classes (cardiolipin – CL,
268
and phosphatidylserine – PS). Similar to the trend for total PLOOH, there are no
270
relative to total phospholipid content. One exception, however, is the 1.5-times increase
272
cold-acclimated fish (Fig. 4B).
274
a muscle type and also between red and white muscles. The rank order of oxidized
276
> PI > PS > CL. In contrast, the ranking of PLOOH for red muscle varies between
278
25°C). These results demonstrate an increased oxidation of CL in red muscle compared
280
oxidative (red) fibers.
273
The rank order of PLOOH by class is different among acclimation groups within
275
phospholipids for white muscle from both 7°C and 25°C acclimated animals is PE > PC
277
acclimation groups (PE > PC > CL > PI > PS for 7°C and PC > PE > CL > PI > PS for
279
with white muscle, which is likely explained by higher contents of mitochondria in
281
Levels of Vitamin E and Ubiquinone
283
altered by temperature acclimation. Vitamin E, CoQ9, and an unidentified ubiquinone
285
mitochondrial membranes of warm-acclimated animals, when compared to cold-
287
Phospholipid Composition
289
groups in membranes from both red and white muscle (Tables 1 and 2). Although
291
other phospholipid classes are identifiable in both membranes, and the majority of these
293
from cold-acclimated fish are enriched in PE, relative to PC. Mitochondrial membranes
295
higher relative to warm-acclimated individuals, while SR membranes from the cold-
297
6A and 6D; t-test; p < 0.05).
299
altered by temperature acclimation in both mitochondria and SR (Tables 3 and 4). Higher
301
raise the unsaturation index (UI) of some phospholipid classes, including PC (SR only)
303
acclimated animals than cold-acclimated fish (Tables 5 and 6). More specifically,
282
Levels of vitamin E and ubiquinone (CoQ) in mitochondrial membranes are
284
homologue (CoQ?) relative to phospholipid contents are 5- to 3-times higher in
286
acclimated counterparts (Fig. 5; t-test; p < 0.05).
288
Temperature acclimation induces significant remodeling of phospholipid head
290
mitochondria and SR are dominated by phospholipids with PE and PC headgroups, nine
292
vary in response to temperature acclimation (Tables 1 and 2). As expected, membranes
294
from cold-acclimated animals have ratios of PE-to-PC that are on average 1.8-times
296
acclimated group show a more modest 1.4-times increase, on average, in this ratio (Fig.
298
Phospholipids varying in contents of saturated and unsaturated fatty acids are also
300
amounts of polyunsaturated fatty acids (PUFA) following cold-acclimation significantly
302
and PE (both membranes), while the UI in PS is higher in both membranes from warm-
304
mitochondrial membranes from red muscle following cold-acclimation have a 1.4-times
306
mitochondria prepared from cold-acclimated animals (Fig. 6B; t-test; p < 0.05), while SR
308
0.0001). While the magnitude of response is less than in red muscle, total UI of both
310
increased by a modest 10%, relative to animals at warm body temperature (Fig. 6E and
312
DISCUSSION
314
and levels of acyl chain unsaturation during thermal acclimation is not sufficient to offset
316
membranes to LPO is not conserved with temperature variation. In fact, patterns of
318
of striped bass (Morone saxatilis). Even with elevated levels of unsaturated fatty acyl
320
LPO normalized to phospholipid content are generally similar (red muscle) or even lower
322
25°C animals. However, employing a different normalization criterion to rates of LPO
324
of LPO and levels of membrane protein do occur with temperature acclimation. These
326
(e.g.,(6) and Table 7 ), and indicate that the choice of normalization criteria can influence
305
higher UI of PE phospholipids, leading to a total increase in UI of 1.2-times in
307
has 1.2- and 1.3-times higher UI for PC and PE, respectively, (Fig. 6C; t-test; p
