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First published online March 27, 2009
Journal of Experimental Biology 212, 1106-1114 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.027888
Mimicking the natural doping of migrant sandpipers in sedentary quails: effects of dietary n-3 fatty acids on muscle membranes and PPAR expression
Biology Department, University of Ottawa, Ottawa, Ontario, Canada
* Author for correspondence (e-mail: jmweber{at}uottawa.ca)
Accepted 27 January 2009
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: lipid metabolism, membrane phospholipid, peroxisome proliferator-activated receptor gene expression, mitochondrial enzyme, sarcoplasmic reticulum, Colinus virginianus
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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; Maillet and Weber,
2007). In preparation for this non-stop flight, body mass is
doubled by feeding frantically on marine invertebrates containing record
amounts of n-3 eicosapentaenoic acid (EPA) and n-3 docosahexaenoic acid (DHA)
(Maillet and Weber, 2006).
This unusual diet causes the rapid incorporation of n-3 fatty acids in tissue
lipids, thereby augmenting unsaturation, and it increases the activities of
oxidative enzymes in muscle. The observed correlations between the abundance
of n-3 fatty acids in muscle lipids and enzyme activities imply an exciting
functional relationship between diet and energy metabolism
(Maillet and Weber, 2007).
Previous studies suggest that EPA and DHA have different effects and do not
only act through changes in membrane composition but also through
membrane-independent mechanisms. However, current evidence for natural doping
is mainly based on indirect support
(Maillet and Weber, 2006;
Maillet and Weber, 2007). The
observed increase in muscle oxidative capacity may not be entirely caused by
diet but by other seasonal effects of migration such as exercise training and
hormonal changes. Here, we carried out controlled laboratory experiments to
focus on the effects of diet and to attempt to investigate the roles of EPA
and DHA independently. Bobwhite quail (Colinus virginianus) was
selected as a model to characterize the mechanisms of action of n-3 fatty
acids because the diet of this domesticated bird can easily be manipulated in
captivity. In previous studies, it was assumed that the fatty acid composition
of total tissue phospholipids reflects the composition of mitochondrial
membranes (Maillet and Weber,
2006; Maillet and Weber,
2007). However, we could find no published data demonstrating that
all membranes are equally affected by diet.
EPA and DHA are well known to influence metabolism in vivo and
in vitro, either by getting incorporated into membranes
(Hulbert et al., 2005) or
through binding to nuclear receptors that regulate gene expression
(Feige et al., 2006). Diet and
in vitro manipulations have been used to alter the fatty acid
composition of phospholipids. The resulting changes in membrane fluidity,
permeability, n-3/n-6 ratio, and local molecular environment play important
roles in modulating the activities of key membrane proteins
(Gerson et al., 2008;
Guderley et al., 2008;
Murphy, 1990;
Stillwell et al., 1997). They
include enzymes from oxidative pathways, ATPases, hormone receptors and ion
channels [carnitine palmitoyl transferase (CPT)
(Guo et al., 2005;
Power and Newsholme, 1997);
citrate synthase (CS) (Miyasaka et al.,
1996); Na+/K+-ATPase; insulin receptor
(Corcoran et al., 2007);
Na+ and Ca2+ channels
(Leaf et al., 2005)]. EPA and
DHA are also natural ligands for peroxisome proliferator-activated receptors
(PPARs), and these transcription factors regulate the expression of genes
orchestrating fundamental aspects of lipid metabolism. Three PPAR isoforms
with distinct tissue distributions and functions have been identified.
PPAR
and β are mostly involved in stimulating fatty acid oxidation
whereas PPAR
modulates lipid storage and adipocyte differentiation
(Berger and Moller, 2002;
Feige et al., 2006).
Membrane changes (Hulbert et al.,
2005) and/or PPAR-induced mitochondrial and peroxisomal
proliferation (Froyland et al.,
1996; Totland et al.,
2000) can stimulate capacity for aerobic metabolism. It is clear
that n-3 fatty acids affect function from organelles to the whole organism,
even though exact mechanisms are still unknown. The physiological response to
n-3 fatty acids is characterized by increases in the activities of Krebs cycle
and β-oxidation enzymes in mammalian muscle
(Power and Newsholme, 1997),
liver (Totland et al., 2000)
adipose tissue (Guo et al.,
2005) and lymphoid tissue
(Miyasaka et al., 1996).
In vivo capacity for endurance exercise is also affected by dietary
fatty acids in mammals (Ayre and Hulbert,
1997), fish (McKenzie et al.,
1997; Wagner et al.,
2004) and birds (Pierce et
al., 2005). However, these whole-organism studies fail to agree
whether n-3 fatty acids have a positive or negative impact on aerobic
performance.
The goals of this study were therefore to use domestic bobwhite quails as a model to determine: (1) whether dietary n-3 fatty acids alone can stimulate capacity for aerobic metabolism in the flight muscle of a non-migratory bird, (2) whether the activation of oxidative enzymes is caused by changes in the composition of membrane phospholipids and/or PPAR gene expression, (3) whether dietary EPA and DHA have different effects and (4) whether phospholipids from total muscle, mitochondria and sarcoplasmic reticulum show the same pattern of fatty acid incorporation from the diet. We anticipate that the doping effects of n-3 fatty acids previously observed in migrant sandpipers can be replicated in non-migratory quails and are mediated by membrane-related as well as PPAR-related mechanisms. We hypothesize that EPA and DHA have different effects on oxidative metabolism and that all membranes are equally affected by the diet.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Dietary treatments and tissue sampling
The natural diet of migrating semipalmated sandpipers
(Maillet and Weber, 2006;
Maillet and Weber, 2007) was
mimicked by supplementing the food of bobwhite quails with oils administered
by gavage. The birds were randomly divided into five groups of eight
individuals: control (corn oil), EPA (oil enriched with n-3
eicosapentaenoate), DHA (oil enriched with n-3 docosahexaenoate), EPA+DHA (EPA
and DHA oils were given on alternate days) and GEM (gemfibrozil, a
hypolipidemic drug and PPAR agonist). EPA and DHA oils were a generous gift
from Ocean Nutrition (Dartmouth, NS, Canada). The oils (0.7 ml
day–1 for 6 weeks) or GEM (5 mg in 0.7 ml of corn oil per day
for 4 days) were administered using a 14 G gavage needle made of stainless
steel. For each animal, gavage was performed daily in 
30 s with minimal
stress because all the birds were habituated to the procedure. The fatty acid
composition of the food and of the oil supplements is presented in
Table 1. After the gavage
period, the animals were euthanized with CO2 followed by cervical
dislocation. Pectoral muscle was rapidly excised by dissection. Approximately
5 g of muscle was freeze-clamped in liquid N2 to measure enzyme
activities and the fatty acid composition of membrane phospholipids.
Approximately 0.5 g of muscle was snap frozen in liquid N2 to
measure PPAR expression. All samples were stored at –80°C for a
maximum of 3 months before analyses.
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Enzymes
Muscle homogenates were prepared in Hepes buffer (40 mmol
l–1, pH 7.3). The activities of citrate synthase (CS; E.C.
2.3.3.1), total carnitine palmitoyl transferase (CPT I+CPT II; E.C. 2.3.1.21),
3-hydroxyacyl CoA dehydrogenase (HOAD; E.C. 1.1.1.35) and cytochrome oxidase
(COX; E.C. 1.9.3.1) were measured at 37°C using a Spectramax
spectrophotometer (Gemini XS, Molecular devices, Sunnyvale, CA, USA) and
96-well flat-bottom microplates (Thermo Fisher Scientific, Nepean, ON,
Canada). Enzyme activity was measured in triplicate and mean values were used
for calculations. Preliminary measurements were performed to determine
homogenate concentrations yielding maximum reaction velocities. Detailed
procedures for enzyme assays have been described previously [CS, CPT and HOAD
(Maillet and Weber, 2007); COX
(Moyes et al., 1997)].
PPAR expression
Partial coding sequences for quail PPAR, β and , as well
as for 18S, were cloned from quail pectoral muscle, and the sequence
information was deposited in GenBank (accession numbers shown in
Table 2). Changes in the
expression of PPAR, β and were measured in pectoral
muscle. All reagents were obtained from Invitrogen (Burlington, ON, Canada)
unless otherwise indicated. Total RNA was isolated from 0.1 g frozen
muscle using TRIzol® reagent (Gibco BRL, Burlington, ON, Canada). RNA
concentration and quality were verified using NanoDrop 1000 (Thermo Fisher
Scientific). Isopropanol and linear acrylamide (Ambion, Austin, TX, USA) were
added to aid RNA precipitation. The samples were placed for 30 min on dry ice
and centrifuged to collect the RNA pellet (12,000g, 5 min,
4°C). Total RNA was DNase-treated (DNase I, Amplification Grade), and
first-strand cDNA synthesis was performed using 1 µg RNA in 11 µl
RNase-/DNase-free water, and primed with 1 µl random hexamer primers. The
mixture was incubated at 65°C for 10 min, quickly chilled on ice, and
briefly spun (13,750 g). Four microliters of 5x reaction
buffer, 2 µl 0.1 mol l–1 DTT, 1 µl 10 mmol
l–1 dNTPs, and 1 µl RNase inhibitor were added, gently
mixed, and the solution was incubated at 42°C for 2 min. One microliter of
SuperScriptTM II RNase H-Reverse Transcriptase or 1 µl of water (NoRT)
was added and the reaction was allowed to continue for 50 min at 42°C. The
reaction was inactivated at 70°C for 15 min and stored at –20°C
until use. The genes of interest were cloned and sequenced, and Primer3
(http://frodo.wi.mit.edu)
was used to design primers based on the gene sequence of bobwhite quail (see
Table 2). Primers of
18–22 nucleotides with optimal annealing temperature between 59 and
61°C were designed to amplify sequences of 150–250 base pairs (bp).
Primers were initially tested using quail muscle cDNA, and the resultant
amplicons were sequenced to confirm specificity.
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Real-time RT-PCR analysis of gene expression was carried out on
first-strand cDNA derived from DNase-treated RNA samples from control and
treatment groups. Each PCR mixture contained 25 ng first-strand cDNA
template, 1x QPCR, 2.5 mmol l–1 MgCl2,
100–400 nmol l–1 gene-specific primer (depending on the
primer set used), 0.25x SYBR green, 200 µmol l–1
dNTPs, 1.25 U HotStarTaq (Qiagen, Mississauga, ON, Canada) and 100 nmol
l–1 ROX reference dye, in a 25 µl total reaction volume.
The primer sets used in this study are reported in
Table 2. Thermal cycling
parameters were as follows: initial 1 cycle Taq activation at 95°C for 10
min, 40 cycles at 95°C for 15 s, 58°C for 5 s, 72°C for 54 s, and
a detection step at 80°C for 22 s. Real-time RT-PCR was assayed on a
MX3005® Multiplex Quantitative PCR system (Stratagene, Mississauga, ON,
Canada) and the accumulation of PCR product was measured in real time as the
increase in SYBR green fluorescence. Data were analyzed using the MxPro
Software Package (Stratagene). The relative expression of the PPAR genes was
normalized to the expression of 18S RNA, which was not affected by the
experimental treatments.
Lipid analysis
The fatty acid composition of membrane phospholipids was measured in total
muscle, isolated mitochondria and isolated sarcoplasmic reticulum.
Chloroform:methanol (2:1 v/v) (Folch et
al., 1957) was used for double extraction of total lipids from 0.5
g muscle homogenized with a Polytron homogenizer (Kinematica, Luzern,
Switzerland) or from 10 mg of isolated mitochondria or isolated sarcoplasmic
reticulum. These organelles were purified by ultracentrifugation as published
previously (Ashour and Hansford,
1983), with the following modifications: 0.5 g of frozen tissue
was used and protein concentration was adjusted to 5 mg
ml–1. After filtration, 0.25% KCl was added and the mixture
was placed at 60°C to separate the organic phase containing the lipids.
This phase was dried on a rotating evaporator (Büchi Rotavapor, Flawil,
Switzerland). The same procedure was used to extract total lipids from the
food and oil supplements. Phospholipids in total muscle, mitochondria or
sarcoplasmic reticulum were separated from total lipids using Supelclean LCNH2
solid-phase extraction tubes (Sigma, St Louis, MO, USA). The fatty acid
composition of the phospholipids was measured using gas chromatography after
transesterification as previously (Maillet
and Weber, 2006). Fatty acid methyl esters were analyzed on an
Agilent Technologies 6890N gas chromatograph (Mississauga, Ontario, Canada)
equipped with a fused silica capillary column (Supelco DB-23, 60 mx0.25
mm i.d., 0.25 µm film thickness) using hydrogen as carrier gas. The system
was equipped with an automatic injection system (Agilent Technologies 7683B
Series). Detailed conditions of analysis have been described previously
(Magnoni and Weber, 2007).
Calculations and statistical analyses
Enzyme activities (µmol min–1 g–1)
were calculated as follows:
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Abs is the change in absorbance at 340, 412 or 550 nm,
t is the reaction time in min, Vf is the
final cuvette volume in µl, Vh is the volume of added
homogenate in µl, 
is the extinction coefficient in
µmol–1 ml (13.6 for DNTB, 6.22 for NADH and 28.5 for
cytochrome c) and D is the dilution factor of the
homogenate. Fatty acids accounting for less than 1% of total fatty acids in
phospholipids were not included in the analysis. Statistical analyses were performed using SYSTAT 8.0 or SigmaStat 3.1 (Systat Software, Chicago, IL, USA). Principal component analysis was used to identify which fatty acids from membrane phospholipids were affected by the treatments. The effects of the diets on enzyme activities (Fig. 1), PPAR gene expression (Fig. 2), % individual fatty acids in membrane phopholipids (Figs 3, 4, 5) and n-3/n-6 ratio (Fig. 7) were analyzed using one way non-parametric ANOVA on ranks and Bonferroni post-hoc test. Non-parametric t-tests on ranks were performed to examine the effects of GEM on PPAR gene expression (Fig. 2) and on the difference in n-3/n-6 ratio between lean and fat semipalmated sandpipers (Fig. 7). Relationships between enzyme activities and % contribution of individual fatty acids in membrane phospholipids (Table 4) or between enzyme activities and n-3/n-6 ratio (Fig. 8) were assessed by linear regression. Statistical significance was set at P<0.05 and all the values presented are means ± s.e.m.
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Effects of dietary n-3 fatty acids and GEM on PPAR gene expression
The effects of EPA, DHA, EPA+DHA and the fibrate drug GEM on the expression
of genes coding for PPAR, β and in pectoral muscle are
reported in Fig. 2. Overall,
the n-3 fatty acid supplements had no significant effects on the relative
expression of any PPAR gene (P>0.05). GEM caused minor, but
statistically significant, 1.5-fold and 2-fold increases in the expression of
PPAR and PPAR (P<0.05), respectively.
Incorporation of dietary n-3 fatty acids in muscle membranes
Table 3 shows the fatty acid
composition of flight muscle membranes and how it is affected by dietary EPA
and DHA. Principal component analysis revealed that only three fatty acids
from membrane phospholipids were modified by the diets; they were arachidonic
acid (ARA), EPA and DHA, and are shown with grey highlights in
Table 3. Dietary supplements
had no significant effect on the relative membrane abundance of saturated,
monounsaturated and polyunsaturated fatty acids (P>0.05). However,
the degree of unsaturation was increased by the EPA and DHA diets
(P<0.05), and the n-3/n-6 ratio was increased by all diets
(P<0.05). The fatty acids whose relative abundance was affected
significantly by the diets in the different membranes from muscle are reported
in Figs 3,
4,
5. For total muscle membranes,
all the diets caused an increase in %EPA and %DHA, as well as a decrease in
%ARA (Fig. 3)
(P<0.05). These dietary effects were the same in mixed membranes
from total muscle tissue (Fig.
3), in membranes isolated from muscle mitochondria
(Fig. 4) and in sarcoplamic
reticulum (Fig. 5)
(P>0.05).
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Changes in membrane composition of bobwhite quails: comparison with refueling semipalmated sandpipers
Figs 6 and
7 compare the changes in
membrane phospholipids observed in sedentary quails fed artificial diets under
controlled conditions (present study) and in wild semipalmated sandpipers
refueling naturally on marine invertebrates just before a long migratory
flight (Maillet and Weber,
2006). The artificial diets of bobwhite quails
(Fig. 6A) and the natural diet
of semipalmated sandpipers (Fig.
6B) had the same effects on flight muscle membrane composition:
increases in %EPA and %DHA accompanied by a decrease in %ARA. However, the
changes in EPA and ARA were quantitatively larger in quails than in
sandpipers. Fig. 7 shows that
the artificial diets of quails and natural diet of sandpipers caused a
significant increase in the n-3/n-6 ratio of muscle membranes
(P<0.05).
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Relationship between enzyme activities and membrane composition
Possible relationships between the relative abundance of variable membrane
fatty acids and oxidative enzyme activities in pectoral muscle were
investigated using linear regression (Table
4). Flight muscle CS activity was positively associated with %EPA,
but no significant relationship was found for %DHA and %ARA. HOAD activity was
positively associated with %EPA and %DHA and was negatively associated with
%ARA. The relationship between %EPA and muscle COX activity had a significant
positive slope. However, %EPA, %DHA and %ARA showed no relationships with CPT
activity. Regression analyses between the n-3/n-6 ratio of membrane
phospholipids and enzyme activities are presented in
Fig. 8. This ratio was
positively associated with CS and HOAD, but no relationship was found for CPT
and COX.
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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; Maillet and
Weber, 2007). On its own, the diet is able to increase the
activities of oxidative enzymes by 58–90% in the flight muscle of a
non-migratory bird (Fig. 1): an
increase in aerobic capacity normally only observed after prolonged endurance
training. We have used a quail model to show that changes in membrane
composition play an important role in mediating the metabolic effects of the
diet. However, results provide no significant support for the involvement of
PPARs as determined by changes in gene expression. All membranes are equally
affected by diet because total tissue phospholipids, mitochondrial membranes
and sarcoplasmic reticulum show the same changes in fatty acid composition.
Presumably, birds can interconvert EPA and DHA since the consumption of either
fatty acid has the same effects on membrane composition. However, EPA and DHA
appear to upregulate oxidative metabolism through different pathways because
their pattern of enzyme stimulation is specific.
Effects of n-3 fatty acids on oxidative enzymes
The stimulation of oxidative capacity by n-3 fatty acids has been
demonstrated in a variety of mammalian tissues including muscle
(Power and Newsholme, 1997),
liver (Totland et al., 2000),
adipose (Guo et al., 2005) and
lymphoids (Miyasaka et al.,
1996). The fatty acid composition of the diet is also known to
affect whole-organism aerobic capacity
(
O2,max) in rats
(Ayre and Hulbert, 1997),
salmon (Wagner et al., 2004)
and one species of migrant bird (Pierce et
al., 2005). Previous studies on semipalmated sandpipers could not
eliminate the possibility that their pre-migration increase in oxidative
capacity could be caused by hormonal changes or exercise training
(Maillet and Weber, 2006;
Maillet and Weber, 2007).
Here, through controlled laboratory experiments, we show that diet alone can
strongly stimulate flux capacity through the Krebs cycle and β-oxidation
in a sedentary bird. Therefore, dietary n-3 fatty acids are responsible for
the metabolic changes observed in refueling sandpipers, not other factors
associated with migration. Six weeks of n-3 fatty acid supplements were
sufficient to increase enzyme activities by 58–90% in quail flight
muscle (Fig. 1). This effect of
the diet is impressive because such a strong response can only be obtained
through prolonged endurance training in mammals. A survey of the literature
shows that aerobic exercise training can stimulate enzyme activities by up to
42% in rats [after 8 weeks of training
(Leandro et al., 2007;
Siu et al., 2003)],
38–76% in humans [7 weeks (Carter et
al., 2001)] and 41–72% in horses [10 weeks
(Kim et al., 2005)].
Therefore, the increases in oxidative enzyme activities observed in birds
eating n-3 fatty acids can surpass those reported for mammals after endurance
training and they occur more rapidly.
The largest increments in enzyme activities reported for training mammals
were smaller than in the present study and they were accompanied by
improvements of 10–26% in mass-specific
O2,max
(Bedford et al., 1979;
Carter et al., 2001;
Kim et al., 2005). Therefore,
dietary n-3 fatty acids were probably able to boost the aerobic capacity of
quails by more than 20%, although their
O2,max could not
be quantified in our study. The effect of diet on enzyme activities was
stronger in quails (Fig. 1)
than in refueling sandpipers (Maillet and
Weber, 2007). Potential reasons for this difference include: (1)
sedentary quails may have more scope for improvement because their baseline
activities are low, (2) the n-3 fatty acid content of the diets were different
(59 vs 31% EPA and 70 vs 14% DHA for quails vs
sandpipers) and (3) increased consumption of n-3 fatty acids lasted longer for
quails than sandpipers (6 vs 2 weeks). The oxidative capacity of
flight muscle is known to be much lower in sedentary than in migrant birds
(Lundgren and Kiessling,
1986), and bobwhite quails are no exception to this pattern.
Surprisingly, the effects of dietary n-3 fatty acids were strong enough to
activate the enzymes of sedentary quails to levels only normally observed in
migrants (Bishop et al., 1995;
Driedzic et al., 1993;
Guglielmo et al., 2002;
Maillet and Weber, 2007).
In one of the experimental treatments, EPA and DHA were administered together. It was selected to mimic the natural diet of semipalmated sandpipers and to test whether birds take advantage of possible synergistic effects. Overall, the combined effects of the two dietary fatty acids do not provide a metabolic advantage, except maybe for COX, whose activity was only increased in the EPA+DHA group (Fig. 1). The variable responses obtained between dietary treatments support the notion that EPA and DHA act through different pathways, and two potential mechanisms of action were investigated: incorporation in membrane phospholipids and activation of PPAR gene expression.
Incorporation of n-3 fatty acids in muscle membranes
The fatty acid composition of membrane phospholipids can be altered by the
diet (Awad, 1986;
Guderley et al., 2008;
Maillet and Weber, 2006), but
no previous study had established whether all membranes respond similarly.
Results reveal that the consumption of n-3 fatty acids modifies all membranes
equally (Figs 3,
4,
5) and, therefore, that
compositional changes of total tissue phospholipids mirror those of
mitochondrial and sarcoplasmic membranes. Consumption of the experimental
diets only caused changes in the abundance of three membrane fatty acids
(increases in %EPA and %DHA were compensated by a decrease in %ARA)
(Table 3; Figs
3,
4,
5). This response closely
mimics the changes in fatty acid composition and n-3/n-6 ratio observed in the
muscle membranes of sandpipers during pre-migration fattening (Figs
6 and
7). Therefore, the bobwhite
quail is a useful experimental model to investigate the mechanisms responsible
for the doping effects of n-3 fatty acids. Although qualitatively identical
between quails and sandpipers, compositional changes were quantitatively
stronger in quails. In future studies, the experimental procedure could be
adjusted by reducing gavage time to less than 6 weeks to obtain exactly the
same changes in composition between quails and sandpipers. Alternatively, the
enhanced membrane response elicited here may amplify the doping mechanisms
under investigation, thereby making them easier to study.
The fatty acid composition of membrane phospholipids can also be altered
through regular exercise, and some of the diet-induced changes found in quails
match those observed in mammals after endurance training. Of particular
interest is the fact that the muscles of humans subjected to 8 weeks of
aerobic training show significant increases in %DHA (+31%) and n-3/n-6 ratio
(+80%) (Andersson et al., 2000;
Helge et al., 2001). By eating
n-3 fatty acids for 6 weeks, quails were able to exaggerate these aspects of
the human training response. They showed remarkable increases of 35–69%
in %DHA and of 240–320% in their n-3/n-6 ratio
(Table 3; Figs
3,
4,
5,
7). The large change in %DHA is
particularly interesting because the membrane abundance of this fatty acid
seems to play a significant role in modulating the sensitivity of CPT to
malonyl-CoA, its natural inhibitor (Morash
et al., 2008).
Quails may have the capacity for EPA–DHA interconversion because the
same changes in membrane composition are observed after the consumption of
either fatty acid (%DHA is increased by eating EPA, and %EPA is increased by
eating DHA) (Figs 3,
4,
5). However, this possibility
should be taken with caution because our DHA diet also contains significant
amounts of EPA, and the EPA diet contains some DHA
(Table 1). Tracer studies have
shown that rats and humans have limited capacity for retroconversion of DHA to
EPA when their diet includes normal DHA levels [only 1.4% of dietary DHA is
retroconverted to EPA (Brossard et al.,
1996)]. By contrast, the pathway is strongly stimulated when
humans consume DHA supplements for 6 weeks [up to 12% of dietary DHA is
retroconverted (Arterburn et al.,
2006; Conquer and Holub,
1997)]. Unfortunately, actual conversion capacities in either
direction have never been measured in birds and this obscures our ability to
distinguish specific effects of EPA or DHA.
Finally, membranes can act as a reservoir for signaling molecules like
anti-inflammatory n-3 fatty acids and pro-inflammatory n-6 fatty acids that
can be recruited to regulate the inflammation response
(Surette, 2008). Therefore,
increasing the n-3/n-6 ratio of membrane phospholipids causes chronic
inhibition of inflammation pathways. In migrant birds, a reduced capacity for
inflammation caused by eating large quantities of n-3 fatty acids could be
greatly beneficial because long-distance flights are known to cause muscle
damage (Guglielmo et al.,
2001).
Relationship between membrane composition and oxidative metabolism
The local molecular environment of proteins affects their function and, for
many enzymes, it can be altered by modulating the fatty acid composition of
membrane phospholipids. Therefore, the changes in membrane fluidity,
permeability and n-3/n-6 ratio that result from n-3 fatty acid incorporation
are known to influence the activities of key oxidative enzymes and ATPases
(CS, CPT, Na+/K+-ATPase and Ca2+-ATPase among
others) (Miyasaka et al.,
1996; Power and Newsholme,
1997; Swanson et al.,
1989; Turner et al.,
2005). In an attempt to explore possible functional links, we have
identified several associations between enzyme activities and particular
characteristics of membrane composition (n-3/n-6 ratio and relative abundance
of long-chain polyunsaturated fatty acids)
(Fig. 8;
Table 4). However, these
regressions should be interpreted with caution because the membrane parameters
selected for this analysis may not reflect compositional characteristics that
really affect enzyme function. Therefore, the absence of significant
relationships cannot be used to eliminate membrane-related mechanisms of
enzyme regulation. Conversely, the presence of a significant regression does
not prove a mechanistic link but merely suggests its possibility. Keeping
these important limitations in mind, we have observed that enzyme activities
were correlated with the n-3/n-6 ratio (CS and HOAD), %EPA (CS, HOAD and COX),
%DHA (HOAD) and %ARA (HOAD) (Fig.
8; Table 4).
Therefore, changes in membrane composition may not only affect enzymes located
within them (such as COX) but matrix enzymes as well. A potential explanation
for this interesting observation is that many so-called `matrix enzymes' are
preferentially placed in very close proximity to inner mitochondrial membranes
(D'Souza and Srere, 1983b) or
are even actually bound to them, e.g. CS
(D'Souza and Srere, 1983a).
The observed correlations are very useful because they allow the design of
further experiments to investigate specific mechanisms of enzyme activation
via changes in membrane composition. Overall, direct substitution of
n-6 ARA by n-3 EPA appears to play the most prominent role in activating the
enzymes examined in this study. The absence of associations between the
activity of some enzymes and membrane composition suggests that
membrane-independent mechanisms of activation could also be at play.
Expression of PPAR genes
PPARs are transcription factors activated by natural and synthetic ligands
such as long-chain polyunsaturated fatty acids and some hypolipidemic drugs
(Feige et al., 2006).
PPAR, β and mRNAs are present in quail flight muscle.
Expression levels were not affected by the consumption of n-3 fatty acids
(Fig. 2) but they did respond
to GEM (a synthetic PPAR agonist). Previous studies had failed to show any
changes in PPAR expression in adult hen livers and in isolated
hepatocytes from chicken embryos after they were treated with the other
fibrate drugs clofibrate and fenofibrate
(Cwinn et al., 2008;
Konig et al., 2007). Reasons
for this discrepancy are unclear but may reflect differences between drugs,
tissues, exposure times or doses. The fact that dietary n-3 fatty acids did
not stimulate the expression of PPAR genes
(Fig. 2) is insufficient
evidence to eliminate the involvement of PPAR-related mechanisms in enzyme
activation. This is because the recruitment of PPAR pathways may only occur
with early activation of gene expression (i.e. earlier than 6 weeks after
starting the diets) or without significant changes in mRNA levels if turnover
is high. In birds, the upregulation of some target genes (e.g. CPT, lipopotein
lipase) can be triggered by fibrate drugs in the absence of changes in the
expression of PPAR genes (Cwinn et al.,
2008; Konig et al.,
2007). More experiments are needed to establish whether
PPAR-related mechanisms are activated by dietary n-3 fatty acids in bird
muscle. Measuring whether PPAR protein expression is stimulated or if PPAR
inhibitors can affect the doping response are promising avenues for future
work.
Conclusions
The changes in membrane composition previously observed in refueling
sandpipers were replicated here in sedentary quails that served as a model to
characterize mechanisms of natural doping. Results show that the substitution
of n-6 ARA by n-3 EPA in membrane phospholipids plays an important role in
mediating the metabolic effects of the diet. EPA and DHA have the same
stimulating effect on oxidative metabolism, possibly because birds can
interconvert the two fatty acids. Changes in the fatty acid composition of
mitochondrial membranes and sarcoplasmic reticulum can be assessed by
monitoring total muscle membranes because all phospholipids are equally
affected by diet. The oxidative capacity of bird muscles is very strongly
stimulated by dietary EPA and DHA and this physiological response occurs
rapidly. Only extreme regimes of endurance training can lead to increments in
oxidative capacity matching those induced here by diet in domestic quails. In
preparation for long migrations, some birds improve their physical fitness by
eating! When maximal energy storage is critical, choosing n-3 fatty acid
doping over endurance training strikes us as the better strategy to increase
aerobic capacity.
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Footnotes |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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