Full text of DOI:10.1007/s11745-000-0625-8

Cold Acclimation or Grapeseed Oil Feeding
Affects Phospholipid Composition and Mitochondrial
Function in Duckling Skeletal Muscle
François Chaînier, Damien Roussel, Bruno Georges, Roger Meister*,
Jean-Louis Rouanet, Claude Duchamp, and Hervé Barré
Laboratoire de Physiologie des Régulations Energétiques, Cellulaires et Moléculaires (Unité Mixte de Recherches
5578 Centre National de la Recherche Scientifique - Université Lyon 1), F-69622 Villeurbanne Cedex, France
ABSTRACT: The phospholipid fatty acid (FA) composition and (2–5). This remarkable adaptation to cold exposure differs
functional properties of skeletal muscle and liver mitochondria
were examined in cold-acclimated (CA, 4°C) ducklings. Phos-
pholipid FA of isolated muscle mitochondria from CA birds
were longer and more unsaturated than those from thermoneu-
tral (TN, 25°C) reared ducklings. The rise in long-chain and
polyunsaturated FA (PUFA, mainly 20:4n-6) was associated
with a higher State 4 respiration rate and a lower respiratory
control ratio (RCR). Hepatic mitochondria, by contrast, were
much less affected by cold acclimation. The cold-induced
changes in phospholipid FA profile and functional properties of
muscle mitochondria were reproduced by giving TN ducklings
from the situation in mammals, as birds lack the specialized
thermogenic brown adipose tissue (BAT) of newborn mam-
mals, hibernators, and small rodents (6–8). In the absence of
BAT, skeletal muscle appears as the main site of cold-induced
NST (9).
Muscle NST may be based on a loose-coupling of mito-
chondrial oxidative phosphorylation controlled by fatty acids
(FA) on the basis of results obtained with isolated mitochon-
dria in cold-acclimated (CA) ducklings (3,10,11). These stud-
ies have contributed to clarify the earlier observation of loose-
a diet enriched in grapeseed oil (GO, rich in n-6 FA), suggest- coupled mitochondria and possible involvement of FA in CA
ing a causal relationship between the membrane structure and
mitochondrial functional parameters. However, hepatic mito-
chondria from ducklings fed the GO diet also showed an en-
richment in long-chain PUFA but opposite changes in their bio-
chemical characteristics (lower State 4, higher RCR). It is sug-
gested that the differential modulation of mitochondrial
functional properties by membrane lipid composition between
skeletal muscle and liver may depend on muscle-specific fac-
tors possibly interacting with long-chain PUFA and affecting the
proton leakiness of mitochondrial membranes.
birds (12,13). The molecular mechanisms of such loose-cou-
pling are still unclear, but involve an increase (+40%) in the
mitochondrial membrane conductance to protons in CA duck-
lings (11). No uncoupling protein with protonophoric proper-
ties similar to those expressed in mammalian tissues has yet
been described in CA birds (7,8). Nevertheless, because phos-
pholipid membranes are inherently leaky to protons (14,15)
and the degree of proton leak correlates with the composition
in unsaturated FA (16,17), a change in the phospholipid com-
position of mitochondrial membrane may contribute to the in-
creased leakiness of mitochondrial membranes in CA duck-
lings. Differences in nonphosphorylating respiratory rates of
isolated mitochondria from various species have already been
related to mitochondrial phospholipid composition (18). Con-
sistent with this idea is the observation that in CA rats, the
membranes of BAT mitochondria contain more polyunsatu-
rated FA (PUFA), possibly contributing to the thermogenic
activation of BAT mitochondria (19). Furthermore, an enrich-
ment of membranes with PUFA obtained through changes in
the lipid composition of the diet was associated with an in-
creased skeletal muscle oxygen consumption (20).
Paper no. L8546 in Lipids 35, 1099–1106 (October 2000).
When chronically exposed to cold, most endotherms are able
to enhance their metabolic capacity to generate heat to com-
pensate for the rise in heat loss and maintain homeothermy.
Small rodents are thus well known to develop nonshivering
thermogenic (NST) capacity in the cold (1). Some species of
birds, including chickens, ducks, and penguins, can also de-
velop NST in response to cold acclimation or acclimatization
*To whom correspondence should be addressed at Laboratoire de Physiolo-
gie des Régulations Energétiques, Cellulaires et Moléculaires, (UMR 5578
CNRS-Université Lyon 1), Faculté des Sciences, 43 bld. du 11 Novembre
1918, bât 404, F-69622 Villeurbanne Cedex, France.
The aim of this study was therefore to assess whether the
development of skeletal muscle NST in CA ducklings was ac-
companied by changes in the FA composition of tissue mem-
branes. In a first experiment with whole tissues, we found that
there was an enrichment in PUFA in skeletal muscle phospho-
lipids of CA ducklings. In a second experiment, we examined
whether the cold-induced changes in phospholipid composi-
E-mail: meister@physio.univ-lyon1.fr
Abbreviations: ANOVA, analysis of variance; BAT, brown adipose tissue;
CA, cold acclimated; FA, fatty acid; FOG, fast-oxidative glycolytic; GO,
grapeseed oil; MUFA, monounsaturated fatty acid; NST, nonshivering ther-
mogenesis; PUFA, polyunsaturated fatty acid; RCR, respiratory control ratio;
SFA, saturated fatty acid; TN, thermoneutral control ducklings; U.A.R.,
Usine d’Alimentation Rationnelle.
Copyright © 2000 by AOCS Press
1099
Lipids, Vol. 35, no. 10 (2000)

1100
F. CHAÎNIER ET AL.
TABLE 1
tion were causally related to the functional characteristics of
isolated mitochondria. To this purpose, mitochondrial mem-
brane FA composition was altered at thermoneutrality by
changing the lipid composition of the diet (21). The conse-
quences of a diet supplemented with grapeseed oil rich in n-6
FA on the phospholipid composition and functional proper-
ties of hepatic and muscle mitochondria were thus examined
in ducklings kept at thermoneutrality and compared to those
observed after cold acclimation.
Fatty Acid Composition (in %) of Diets
Genthon 5A
UAR 115
GO diet
14:0
14:1n-7
16:0
16:1n-9
16:1n-7
18:0
18:1n-9
18:1n-7
18:2n-6
20:0
20:1n-9
20:2n-6
20:3n-6
22:0
20:4n-6
20:5n-3
24:0
24:1n-9
22:5n-3
22:6n-3
3.49
0.24
15.68
0.26
1.13
5.76
27.98
1.30
37.98
0.41
2.28
0.15
0.19
—
0.05
—
0.10
—
—
0.20
0.31
0.06
11.39
0.07
1.00
2.78
22.02
1.41
49.61
0.41
5.10
0.19
0.05
0.04
0.17
0.67
0.28
0.20
0.12
1.38
0.15
0.03
8.46
0.04
0.42
3.37
20.28
1.00
60.52
0.30
2.21
0.12
0.07
0.05
0.07
0.26
0.17
0.06
—
MATERIALS AND METHODS
Animals. Animals were cared for under the French Code of
Practice for the Care and Use of Animals for Scientific Pur-
poses, and the experimental protocols were approved by the
French Ministry of Agriculture Ethics Committee (section an-
imals). Male Muscovy ducklings (Cairina moschata L., pedi-
gree R51, Institut National de la Recherche Agronomique,
France) were obtained from a commercial stockbreeder (Ets
Grimaud, France). They were fed ad libitum with a commer-
cial mash (Genthon 5A; Genthon, Cheyssieu, France, see
Table 1) and had free access to water. From 1 wk of age,
ducklings were assigned to experimental groups.
A first batch of ducklings was divided into two groups; one
was kept at 25°C and constituted the thermoneutral (TN) con-
trol group, while the other one was reared in the cold (4°C)
and constituted the CA group. These birds were kept on the
Genthon 5A commercial mash. This cold-acclimation sched-
ule was shown to stimulate the development of skeletal mus-
cle NST by 5 wk of age (6,9). Ducklings were killed by de-
capitation and tissues (gastrocnemius muscle and liver) were
sampled and frozen in liquid nitrogen and kept at −70°C until
FA analysis of total tissue phospholipids.
Following the results of the first experiment, a second
batch of ducklings was divided into three experimental
groups. Two groups of TN or CA ducklings were fed ad libi-
tum with a commercial mash [UAR 115; Usine d’Alimenta-
tion Rationnelle (U.A.R.), Villemoisson, France]. A third
group of ducklings kept at thermoneutrality was fed with the
UAR 115 mash supplemented with 6.5% (w/w) grapeseed oil
(GO; U.A.R.). Composition and energy content of diets are
presented in Table 1. By 5 wk of age, ducklings were killed
by decapitation. As the CA group grew a little bit more slowly
(Fig. 1), they were killed a few days later at the same body
weight as the TN and GO groups. Red internal gastrocnemius,
rich in slow-oxidative and fast-oxidative glycolytic (FOG)
fibers, white external gastrocnemius, rich in fast glycolytic
and FOG fibers (22), and liver samples were taken and used
for the analysis of mitochondrial function and FA com-
position of mitochondrial phospholipids. Photoperiod was
8 h/16 h (light/dark). Food consumption and body weight of
ducklings were measured daily.
0.54
Total SFA
Total MUFA
Total PUFA
Total n-6
Total n-3
n-3/n-6
25.24
33.78
39.25
38.64
0.92
15.21
29.86
52.19
50.02
2.17
12.50
24.04
61.58
60.78
0.80
0.02
0.04
0.01
Unsaturation indexa
Mean chain lengthb
Lipid content (%)
1.14
17.08
4.5
1.42
17.47
3.5
1.50
17.57
9.5
Energetic valuec (kcal/g)
4.20
3.38
3.74
aUnsaturation index = (∑mi·ni)/100, where mi is the mole percentage and ni
is the number of C–C double bonds of the fatty acid i.
bMean chain length index = (∑mi ·ni)/100, where mi is the mole percentage
and ni is the number of C of the fatty acid i.
cEnergetic value has been calculated according to 4 kcal/g for digestible pro-
teins and carbohydrates and 9 kcal/g for digestible lipids. GO, grapeseed oil;
SFA, saturated fatty acid; MUFA, monounsaturated fatty acid; PUFA, polyun-
saturated fatty acid.
Isolation of skeletal muscle mitochondria. Internal and ex-
ternal gastrocnemius muscle and liver samples were rapidly
taken out, freed of fat and connective tissues, and mixed in a
cold isolation medium with a Teflon glass homogenizer. Mus-
FIG. 1. Effects of grapeseed oil (GO) feeding or cold acclimation on
growth rate. GO ducklings reared at thermoneutrality (▲) and CA duck-
lings (■) are compared with thermoneutral controls (■). Values are
means SE (n = 7).
Lipids, Vol. 35, no. 10 (2000)

COLD, DIETARY FAT AND DUCKLING MITOCHONDRIA
1101
cle intermyofibrillar mitochondria and liver mitochondria identified by comparison with commercially available stan-
were isolated as described previously (10,11).
dards (Supelco, Bellefonte, PA).
Lipid analysis. Tissue and mitochondrial total lipids were
Mitochondrial respiration and enzymatic activities. The
extracted according to Folch et al. (23). Residual water of the respiration of isolated mitochondria (0.5 mg mitochondrial
extracts was removed by oil-vacuum. The lipid extracts were protein/mL) was determined polarographically with a Clark
weighed, diluted in chloroform, and stored at −20°C until use. oxygen electrode (oxygraph Gilson 5/6 H), in a glass cell of
Phospholipids and triglycerides from tissue extracts were 1.5 mL volume, thermostated at 25°C as described previously
separated by thin-layer chromatography on silica gel (G60 (10,11). The ATP synthesis of mitochondria was determined
plates) using diisopropyl ether as migration solvent. Phospho- by the bioluminescence procedure of Wibom et al. (26) at
lipids were recovered by scratching the gel and then methyl- 25°C with some modifications as described previously (11).
ating according to Slover and Lanza (24) before FA analysis. The cytochrome oxidase activity of isolated mitochondria
The FA analysis of phospholipids from mitochondrial was determined polarographically as described elsewhere
extracts was performed with total lipids because the presence (10). As the protein/lipid ratio remained unchanged in all the
of tri- and diglycerides was found negligible, and only traces experiments (data not shown), the specific activity of the en-
of cholesterol were detected. The FA of total lipids were zymes was expressed per mg of protein.
therefore assumed to be those of phospholipids. Methylation
was performed according to Lepage and Roy (25) using SE. One-way or two-way analysis of variance (ANOVA) and
acetylchloride. post-ANOVA Fisher PLSD tests and Student’s t tests were
Statistics and chemicals. Data are presented as means
FA methyl esters were stored in hexane (high-performance used to determine significant differences between groups. Sta-
liquid chromatography grade). Gas–liquid chromatography tistical difference was accepted at P < 0.05.
of FA methyl esters was performed using a Chrompack
Solvent of analytical grade for lipids extraction and analy-
CP9001 chromatograph (Chrompack, Middelburg, The sis was from SDS (Peypin, France); the other chemicals were
Netherlands) equipped with a 50-m capillary column (CP-Sil purchased from Sigma.
88, 0.25 mm internal diameter) and Chrompack Maestro2 in-
tegrator software. The split injector was at 270°C and the
flame-ionization detector at 260°C. Temperature program was
RESULTS
150°C for 8 min, rising to 185°C by 10°C/min, 185°C for 10 Experiment 1: Effect of cold acclimation on the FA composi-
min, rising to 200°C by 10°C/min, and then 200°C for 20 tion of tissue phospholipids. At the time of killing, TN duck-
min. Nitrogen was used as carrier gas (100 kPa). FA were lings were slightly heavier than CA birds (1.43 0.03 vs. 1.20
TABLE 2
Fatty Acid Composition (in %) of Total Phospholipids from Gastrocnemius Muscle
and Liver in Thermoneutral (TN) or Cold-Acclimated (CA) Ducklingsa
Gastrocnemius muscle
Liver
TN
CA
TN
CA
16:0
16:1n-7
18:0
12.6 0.3
—
11.9 0.2*
—
23.6 0.4
0.4 0.1
18.4 0.3
11.3 0.4
1.0 0.1
8.4 0.6
2.4 0.2
21.1 0.6
1.8 0.1
3.6 0.2
0.5 0.1
1.9 0.1
21.2 1.3*
0.7 0.1*
19.3 0.4
13.6 1.0*
1.2 0.1
17.5 0.4
19.8 0.7
0.7 0.1
16.0 0.4
1.5 0.1
11.2 0.4
3.9 0.2
2.9 0.1
1.4 0.1
1.5 0.1
19.3 0.1*
16.6 0.4*
0.5 0.1
15.5 0.3
1.1 0.1*
14.2 0.3*
4.1 0.1
3.1 0.1
1.5 0.2
2.4 0.2*
18:1n-9
18:1n-7
18:2n-6
20:3n-6
20:4n-6
22:4n-6
22:5n-6
22:5n-3
22:6n-3
9.1 1.2
2.7 0.5
19.3 2.9
1.3 0.1*
2.7 0.2*
0.8 0.1*
1.9 0.3
Total SFA
Total MUFA
Total PUFA
Total n-6
Total n-3
n-3/n-6
31.9 0.6
20.7 0.7
39.4 0.9
36.0 0.7
3.3 0.2
33.2 0.2*
17.3 0.4*
43.0 0.3*
38.7 0.2*
4.4 0.1*
42.4 0.3
13.9 0.5
42.2 0.4
37.3 0.6
3.1 0.3
40.9 0.9
16.7 0.9*
41.0 1.7
35.5 1.4
3.5 0.3
0.092 0.004 0.113 0.004* 0.082 0.006 0.097 0.007
Unsaturation index
1.51 0.04 1.66 0.01* 1.70 0.02 1.65 0.09
aValues are mean SE from six (gastrocnemius) or five (liver) animals kept on the Genthon 5A
diet. *P < 0.05 vs. TN. Fatty acids representing a small proportion of the total are not shown but
values are included when possible in the totals presented. See Table 1 for abbreviations.
Lipids, Vol. 35, no. 10 (2000)

1102
F. CHAÎNIER ET AL.
0.03 kg, P < 0.05) despite a lower food intake by 5 wk of and their growth rate lower than those of the ducklings kept
age (106 3 vs. 139 4 kcal·kg−0.75·d−1). The FA profile of at thermoneutrality.
tissue phospholipids (Table 2) differed between the two tis-
Experiment 2: Effect of GO diet or cold on the FA profile
sues, the gastrocnemius muscle being poorer in saturated fatty of mitochondrial phospholipids. Whatever the diet or cold-
acids (SFA) but richer in monounsaturated fatty acids acclimation status, the FA composition of mitochondrial
(MUFA) than the liver. In the gastrocnemius muscle, cold ac- phospholipids differed between tissues (Table 3). As seen in
climation led to a marked increase in the proportion of total the composition of tissue phospholipids, there were more SFA
PUFA and to a lesser extent of SFA at the expense of total and less PUFA in the phospholipids of mitochondria isolated
MUFA. The cold-induced rise in total PUFA was mainly ac- from liver than from gastrocnemius muscles. Mitochondrial
counted for by 20:4n-6, and to a lesser extent by 22:4n-6 and phospholipids from the red internal part of the gastrocnemius
22:6n-3. The unsaturation index of total phospholipids was muscle were richer in PUFA than those from the white part.
thus higher in skeletal muscle from CA than from TN duck- Despite these differences, the unsaturation indexes were sim-
lings. In liver, by contrast, the cold-induced rise in long-chain ilar in the three tissues studied, mainly because of the differ-
PUFA was not observed, and there was only a slight increase ences in the pattern of n-6 FA. The content in 20:4n-6 was
in the proportion of total MUFA mainly accounted for by higher in hepatic than in muscle mitochondria, while the
18:1n-9 FA in CA ducklings. The unsaturation index of liver 18:2n-6 was the most abundant FA in muscle mitochondria.
phospholipids was similar between TN and CA ducklings.
The n-3/n-6 ratio was similar in the three tissues.
Experiment 2: Effect of GO diet or cold on food intake and
The mitochondrial FA profile was differentially affected
growth rate. Despite differences in the energy content of the by cold acclimation in liver and skeletal muscles. In both
diet, TN and GO ducklings had a similar energy intake by 5 parts of the gastrocnemius muscle, a higher unsaturation
wk of age (441 3 and 439 12 kcal·kg−0.75·d−1, respec- index and a longer mean chain length were observed in CA
tively) and grew at similar rates (Fig. 1). Again, the energy ducklings. These parameters reflected the increased propor-
intake of CA ducklings was higher (496 5 kcal·kg−0.75·d−1) tion of PUFA (especially 20:4n-6, and to a lesser extent
TABLE 3
Fatty Acid Composition (in %) of Total Phospholipids from Mitochondria of Internal, External Gastrocnemius Muscle,
and Liver in TN, CA, and GO-Supplemented Ducklingsa
Internal gastrocnemius
External gastrocnemius
CA
Liver
CA
TN
CA
GO
TN
2.6 0.4
GO
TN
GO
14:1n-7
16:0
16:1n-9
16:1n-7
18:0
18:1n-9
18:1n-7
18:2n-6
20:0
20:1 + 18:3n-3
20:2n-6
22:0
20:4n-6
20:5n-3
22:4n-6
22:5n-6
22:5n-3
22:6n-3
1.2 0.1
1.5 0.3
1.1 0.2
2.5 0.4
2.0 0.3
0.1 0.0
0.1 0.0
19.3 0.6
0.3 0.0
0.9 0.1a
0.1 0.0
17.9 0.5
0.3 0.03
0.3 0.0b
10.5 0.3a 8.2 0.2b 8.8 0.2b 12.2 0.4a
9.5 0.3b 10.9 0.4c 19.0 0.8
0.2 0.0
0.1 0.0
0.2 0.0
0.3 0.1
0.2 0.0
0.3 0.0
0.4 0.0
0.4 0.0a 0.3 0.0b 0.2 0.0c
0.6 0.1a
0.4 0.0b
0.2 0.0c
0.9 0.1a
19.1 0.4a 21.7 0.2b 21.5 0.3b 15.1 0.3a 18.4 0.2b 17.2 0.3c 16.2 0.3a 17.0 0.2a 18.9 0.3b
13.1 0.5a 10.8 0.3b 8.2 0.3c 16.5 0.6a 13.8 0.4b 10.7 0.3c 15.2 0.4a 16.9 0.6b
3.5 0.1a 3.7 0.1a 2.6 0.1b 3.7 0.1a 4.2 0.1b 2.6 0.1c 1.8 0.1a 1.6 0.1a
8.7 0.5c
1.1 0.1b
20.2 0.2a 19.4 0.7a 23.9 0.8b 20.7 0.3a 20.1 0.9a 25.7 1.0b 10.1 0.3a
9.0 0.3b 15.2 0.4c
0.3 0.0a 0.2 0.0b 0.3 0.0c
0.6 0.0a 0.6 0.0a 0.4 0.0b
0.2 0.0a,b 0.2 0.0a
0.3 0.0b
0.4 0.0b
1.1 0.1b
0.6 0.1b
0.1 0.0
0.4 0.0a
0.4 0.0a
2.1 0.2
0.1 0.0
0.4 0.0a
0.3 0.0a
1.8 0.2
0.1 0.0
0.3 0.0b
1.0 0.1b
1.4 0.3
0.6 0.0a
0.5 0.0a
0.9 0.1a
0.6 0.0a
0.4 0.0a
0.7 0.1b
0.5 0.0a 0.4 0.0b 1.0 0.0c
0.8 0.0a 0.6 0.1b 0.5 0.0b
12.4 0.5a 16.3 0.7b 15.7 0.6b
1.0 0.1a 0.6 0.0b 0.3 0.0c
1.9 0.1a 2.5 0.1b 2.8 0.1c
9.4 0.4a 12.2 0.4b 12.1 0.6b 18.1 0.4a 17.9 0.7a 20.8 0.6b
1.0 0.0a
1.8 0.1a
1.2 0.1a
1.4 0.1
6.3 0.4a
0.7 0.0b
2.5 0.1b
1.3 0.1a
1.4 0.1
7.0 0.3a
0.4 0.0c
2.9 0.2b
2.3 0.2b
1.2 0.1
4.9 0.4b
0.7 0.1a
1.4 0.1a
1.6 0.1a
0.6 0.0
6.4 0.4
0.6 0.1a
1.5 0.1a
1.3 0.2a
0.5 0.0
6.4 0.4
0.2 0.0b
2.2 0.1b
3.0 0.1b
0.5 0.0
5.6 0.4
1.2 0.1a 1.2 0.1a
1.3 0.1a 1.2 0.1a
6.0 0.3a 6.5 0.2a
2.1 0.1b
1.0 0.1b
4.7 0.3b
Total SFA
Total MUFA
Total PUFA
Total n-6
Total n-3
n-3/n-6
31.0 0.4 31.0 0.2 31.4 0.3
28.6 0.3
29.0 0.2
29.2 0.2
37.8 0.5
38.5 0.4
38.5 0.4
18.6 0.7a 16.6 0.4b 12.4 0.3c 23.9 0.9a 21.3 0.4b 15.8 0.3c 18.6 0.6a 20.0 0.6a 10.5 0.6b
44.7 0.6a 48.2 0.5b 51.7 0.3c 42.4 0.8a 45.9 0.4b 50.8 0.8c 39.3 0.7a 37.8 0.9a 48.6 0.8b
34.4 0.5a 37.5 0.5b 42.9 0.3c 31.9 0.4a 34.3 0.5b 41.4 0.5c 30.3 0.4a 28.8 0.6a 40.1 0.6b
8.3 0.4a 8.3 0.2a
6.0 0.3b
8.7 0.5a
9.1 0.4a
6.5 0.5b
7.7 0.4a
7.5 0.3a
6.3 0.4b
0.24 0.01a 0.22 0.01a 0.14 0.01b 0.27 0.02a 0.27 0.01a 0.16 0.01b 0.25 0.0a 0.26 0.01a 0.16 0.01b
Unsaturation index 1.71 0.03a 1.86 0.02b 1.82 0.03b 1.67 0.04a 1.81 0.02b 1.79 0.05b 1.70 0.03a 1.67 0.04a 1.87 0.03b
Mean chain length 18.4 0.0a 18.5 0.0b 18.5 0.0b 18.2 0.1a 18.4 0.0b 18.4 0.1b 18.4 0.0a 18.4 0.0a 18.5 0.1b
aValues are means SE (n = 7). FA representing less than 0.4% are not shown, but are taken into account for the calculation of the totals. Each tissue value
with a different roman superscript is significantly different (P < 0.05), two-way analysis of variance (ANOVA) and Fisher post-ANOVA test. See Tables 1 and
2 for abbreviations.
Lipids, Vol. 35, no. 10 (2000)

COLD, DIETARY FAT AND DUCKLING MITOCHONDRIA
1103
22:4n-6). The proportion of 22:6n-3 was not significantly in- muscle mitochondria (Table 4), especially in the red part of
creased in mitochondrial phospholipids. The rise in long- gastrocnemius, but not those of liver mitochondria (Table 5).
chain PUFA was somehow compensated for by a decrease in In both parts of the gastrocnemius muscle, there was a higher
the proportion of MUFA (especially 18:1n-9) and an increase State 4 respiratory rate, and consequently a lower respiratory
in the mean chain length of SFA (rise in 18:0 and drop in control ratio (RCR) in CA than in TN ducklings. The ADP/O
16:0). In liver, by contrast, the rise in long-chain PUFA was ratio was, however, not affected by cold acclimation. The spe-
not observed, and cold acclimation induced only minor cific (per mg mitochondrial protein) ATP synthesis was
changes in mitochondrial FA profile, i.e., a slight increase in higher in muscle mitochondria from CA ducklings, but the
18:1, compensated for by a decrease in 18:2n-6. The n-3/n-6 specific cytochrome oxidase activity was unaffected. Because
ratio was not affected by cold acclimation in the three tissues. of a higher amount of mitochondrial proteins in skeletal mus-
Contrary to cold acclimation, the GO diet at thermoneutral- cles of CA ducklings, the total oxidative capacity and ATP
ity affected the three tissues studied similarly and induced a production capacity per organ were increased by cold accli-
nonspecific increase in the proportion of all n-6 phospholipid mation in both skeletal muscles.
FA. In muscle mitochondria, these increases were at least up
The GO diet affected the functional characteristics of he-
to the levels observed in CA ducklings. Consequently, in mus- patic and skeletal muscle mitochondria differently. In liver,
cle mitochondria of GO ducklings, the mean chain length and the GO diet led to a higher ADP/O ratio and a higher RCR
the unsaturation index reached the values observed in CA due to a lower respiratory State 4 as compared with ducklings
ducklings, while in hepatic mitochondria, these parameters fed the standard diet. The other parameters remained un-
were higher than in both TN and CA ducklings. In the three changed. In both parts of the gastrocnemius muscle, the GO
tissues of GO ducklings, the increased proportion of n-6 FA diet induced a lower RCR due to a higher respiratory State 4
was compensated for by a decreased proportion of MUFA and than in control TN ducklings. The specific ATP synthesis ac-
the n-3/n-6 ratio was markedly decreased.
tivity was intermediate between that of TN and CA ducklings,
Experiment 2: Effect of cold acclimation or GO diet on the but because of the increased mitochondrial amount, the total
biochemical characteristics of isolated mitochondria. Cold ATP production per gram of muscle reached the level ob-
acclimation affected the functional characteristics of skeletal served after cold acclimation. Taken together, present results
TABLE 4
Respiratory and Functional Parameters of Mitochondria from Internal and External Gastrocnemius Muscle
in TN, CA, and GO-Supplemented Ducklingsa
Internal gastrocnemiusb
CA
External gastrocnemiusb
CA
Pc
Effect
of
treatment
Effect
Interaction
of muscle treatment ×
type
TN
23.0 1.8
GO
TN
GO
muscle type
State 4
State 3
RCR
26.8 1.0
26.7 1.9
21.8 1.0
26.4 1.6
25.0 1.0 <0.05$,£
ns
5.05 0.14 4.38 0.15 4.63 0.17 5.62 0.31 5.12 0.27 5.14 0.17 <0.05$,£
NS
NS
<0.01
<0.01
NS
NS
NS
NS
116.5 10.0 116.9 3.1 122.3 4.9 121.8 6.2 133.6 6.2 128.1 5.2
ADP/O
1.19 0.04 1.15 0.04 1.26 0.02 1.24 0.03 1.30 0.05 1.31 0.02
NS
Cytochrome-oxidase
specific activity
0.88 0.02 0.88 0.03 0.85 0.02 0.80 0.03 0.80 0.03 0.81 0.03
NS
<0.05
NS
Total cytochrome
oxidase activity
53.2 2.5
64.8 6.6
63.9 4.3
75.5 4.5
78.9 5.6
34.5 2.5
43.2 3.1
42.5 3.2
53.1 3.4
38.3 2.6 <0.05$,£ <0.0001
48.0 4.4 <0.05$,£ <0.0001
NS
NS
NS
NS
Mitochondrial proteins 60.8 2.9 74.06 7.1
Specific ATP-synthesis 66.7 6.5 85.2 2.4
Total ATP production
84.1 7.5 100.9 8.6
92.2 4.5
<0.05$
<0.01
<0.05
4.16 0.56 6.58 0.86 6.00 0.60 3.57 0.34 5.42 0.50 4.47 0.51 <0.01$,£
aThe respiratory reaction medium contained 200 mM sucrose, 5 mM KH2PO4, and 20 mM Tris-HCl, pH 7.4 with a final fatty-acid-free bovine serum albu-
min concentration of 2 mg/mL (0.2% wt/vol). The controlled state of respiration (State 4) was initiated by the addition of 5 mM succinate (sodium salt) in the
presence of rotenone (5 µM) and the active state of respiration (state 3) was initiated by the addition of 100 µM ADP. The method of Estabrook (44) was used
for the calculation of State 4 and state 3 respiration and the respiratory control ratio (RCR). The latter respiratory parameter is a measure of the degree of con-
trol imposed on oxidation by phosphorylation. ADP/O ratio was calculated by using the total oxygen consumed during phosphorylation of a pulse of ADP
added to initiate state 3 respiration.
bValues are means SE. State 4 and State 3 in nmol O/min/mg protein, ADP/O in nmol ADP/nmol O, cytochrome-oxidase specific activity in µmol O/min/mg
protein, total cytochrome-oxidase activity in µmol O/min/g muscle, mitochondrial proteins in mg protein/g muscle, specific ATP-synthesis in pmol
ATP/min/µg protein, and total ATP production in µmol ATP/min/g muscle.
cValues of two-way ANOVA. $Significant difference between CA and TN; £significant difference between GO and TN; NS, nonsignificant (P > 0.05). See Ta-
bles 1, 2, and 3 for other abbreviations.
Lipids, Vol. 35, no. 10 (2000)

1104
F. CHAÎNIER ET AL.
TABLE 5
Respiratory and Functional Parameters of Mitochondria Liver
in TN, CA, and GO-Supplemented Ducklingsa
Liver
CA
TN
GO
State 4
State 3
RCR
ADP/O
11.4 1.0a,b
51.3 4.9
12.3 0.7a
53.5 2.5
9.0 0.9b
48.4 3.4
4.49 0.28a
1.39 0.02a
0.42 0.04
81.1 8.3
4.37 0.09a
1.44 0.02a
0.39 0.02
85.5 4.9
5.47 0.33b
1.52 0.02b
0.31 0.04
66.9 5.3
Cytochrome-oxidase specific activity
Total cytochrome-oxidase activity
Mitochondrial proteins
Specific ATP-synthesis
Total ATP production
195.2 19.2
29.4 3.0
5.67 0.69
217.4 6.4
31.6 4.0
6.84 0.82
222.3 14.3
27.5 3.0
5.31 0.61
aValues are means SE (n = 7). State 4 and State 3 in nmol O/min/mg protein, ADP/O in nmol
ADP/nmol O, cytochrome-oxidase specific activity in µmol O/min/mg protein, total cytochrome-oxi-
dase activity in µmol O/min/g muscle, mitochondrial proteins in mg protein/g muscle, specific ATP-
synthesis in pmol ATP/min/µg protein and total ATP production in µmol ATP/min/g muscle. Each tis-
sue means with a different roman superscript is significantly different (P < 0.05), one-way ANOVA
and Fisher post-ANOVA test. See Tables 1, 2, and 3 for abbreviations.
showed that in skeletal muscles, but not in liver, the GO diet By contrast, there was a decrease in the proportion of total
at thermoneutrality globally mimicked the changes in the mi- unsaturated FA in heart mitochondria from CA rats (28),
tochondrial lipid composition and biochemical parameters in- mainly resulting from a rise in the proportion of 18:0 and a
duced by cold acclimation.
decrease in that of 18:1. No marked changes in the unsatura-
tion index of phospholipid FA have been reported in hepatic
mitochondria of CA rodents (29). These results therefore in-
dicate a tissue-specific modulation of the FA composition of
DISCUSSION
Two main results emerged from the present study. First, CA membrane phospholipids toward an enrichment in long-chain
ducklings showed alterations in the lipid composition of PUFA in the thermogenic organs (BAT or skeletal muscles)
skeletal muscle membranes leading to an increased propor- of endotherms. Such enrichment in long-chain PUFA in mi-
tion of long-chain PUFA. Second, the enrichment of mito- tochondrial membranes of CA ducklings suggests specific
chondrial phospholipids with n-6 PUFA, created by giving roles of these FA in the biophysical properties of these mem-
ducklings kept at thermoneutrality a diet rich in GO, partly branes. It is indeed generally accepted that changes in FA pro-
reproduced the alterations in the functional properties of file of biological membranes alter the fluidity, leakiness to
skeletal muscle mitochondria observed in CA ducklings.
protons, and may contribute to the modulation of the activity
Present results clearly show that cold acclimation altered of membrane-bound enzymes (30–33).
the FA composition of tissue and mitochondrial phospho-
The FA composition of mitochondrial phospholipids could
lipids in the gastrocnemius muscle. Indeed, there was a higher be altered by giving ducklings kept at thermoneutrality a diet
proportion of the longer and more unsaturated FA (mainly rich in GO, providing more n-6 PUFA. This diet composition,
20:4n-6) in tissue and mitochondrial phospholipids from gas- rich in 18:2n-6, was chosen in order to provide ducklings with
trocnemius muscle of CA than in those of TN ducklings re- the FA that were the most affected by cold acclimation as
sulting in increased (+10%) unsaturation index and mean shown in the first experiment with total tissue phospholipids
chain length of membrane FA. By contrast, these changes in (Table 2). Results of experiment 2 indicate that in birds, as in
FA profile were not observed in liver mitochondria. We do mammals (21), the mitochondrial membrane FA composition
not favor the possibility that the difference between liver and can be manipulated by the FA composition of the diet. How-
skeletal muscle is related to the fact that these tissues experi- ever, the enrichment in membrane PUFA is lower than what
ence different temperatures during cold acclimation. Al- could be expected from the diet composition, indicating some
though living at low temperature indeed results in increased homeostatic control of the membrane lipid composition al-
unsaturation in ectotherms (see Ref. 27 for a recent review), ready noted by others (34). Because the diet similarly affected
the difference between liver and skeletal muscle temperature the FA profile of mitochondrial phospholipids of the three tis-
is likely to be small in endothermic ducklings and would not sues investigated, it follows that the tissue-specific changes
exceed a few degrees. This is far less than the thermal chal- observed after cold acclimation are precisely regulated and,
lenge used to induce phospholipid changes in ectotherms. A for instance, are not dependent on the higher food intake or
similar enrichment in PUFA resulting in an increase in the alterations in the supply of blood lipids (35). These changes
total FA unsaturation index of mitochondrial phospholipids may therefore reflect active processes controlling the incor-
has been described in the thermogenic BAT of rodents (19). poration of specific long-chain highly unsaturated FA into
Lipids, Vol. 35, no. 10 (2000)

COLD, DIETARY FAT AND DUCKLING MITOCHONDRIA
1105
membranes for particular metabolic and thermogenic pur- length and the degree of unsaturation of membrane FA (42).
poses. Thyroid hormones are good candidates for the modu- The GO diet had less clear-cut effects on the ATP synthesis,
lation of phospholipid composition in CA ducklings. Indeed, despite marked changes in membrane PUFA, possibly be-
they are known to increase 20:4n-6 and decrease 18:2n-6 cause the proportion of the 18:2n-6 was also markedly in-
(36,37) and concomitantly increase the unsaturation index creased. In rats, the ATP-synthase activity was indeed shown
(38). Furthermore, they are suspected to play a role in the de- to be inversely related to the content in 18:2n-6 (43). A spe-
velopment of NST in birds (4,5).
cific role of the 20:4n-6 or longer n-6 FA in modulating these
Cold acclimation as well as GO feeding, both leading to activities remains to be clarified.
an enrichment of mitochondrial phospholipids in n-6 FA, sim-
In conclusion, this study showed that in CA young birds,
ilarly altered the functional properties of isolated skeletal there is a muscle-specific enrichment of mitochondrial phos-
muscle mitochondria. In both cases, there was an increase in pholipids in long and unsaturated FA. Similar changes do not
State 4 respiration rates and a decrease in respiratory control, occur in liver mitochondria. Feeding a GO diet rich in n-6
indicating a lower degree of control imposed on oxidation by PUFA induced similar changes in lipid composition and mi-
the phosphorylation. Similar loose-coupling of muscle mito- tochondrial functional activity in skeletal muscles as did cold
chondria in CA ducklings has already been reported (11,22) acclimation. It is suggested that the FA composition of mito-
and has been related to an increase in mitochondrial mem- chondrial membranes may contribute to the increased proton
brane conductance to protons (11). The present results sup- leakiness of muscle mitochondria observed in CA ducklings,
port the hypothesis that the FA composition of ducklings’ mi- but that muscle-specific factors control this effect.
tochondrial membranes may contribute, at least in part, to an
increased proton leak across mitochondrial inner membrane.
In mammals as well, the degree of proton leak is correlated
with the composition in unsaturated FA (16,17) and differ-
ences in nonphosphorylating respiratory rates of isolated mi-
(CNRS). François Chaînier was in receipt of a Ministère de l’Edu-
tochondria from various species are related to mitochondrial
ACKNOWLEDGMENTS
This work was supported by a grant from the Université Claude
Bernard, and the Centre National de la Recherche Scientifique
cation National, de l’Enseignement Supérieur et de la Recherche fel-
phospholipid composition (18). However, the PUFA compo- lowship.
sition of mitochondrial membranes may not be the only fac-
tor responsible for the altered coupling of muscle mitochon-
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