Martin et al., 2012

1 23

Journal of Comparative Physiology BBiochemical, Systems, andEnvironmental Physiology ISSN 0174-1578 J Comp Physiol BDOI 10.1007/s00360-012-0712-5

Dietary lipid quality and mitochondrialmembrane composition in trout: responsesof membrane enzymes and oxidativecapacities

N. Martin, D. P. Bureau, Y. Marty,E. Kraffe & H. Guderley

1 23

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ORIGINAL PAPER

Dietary lipid quality and mitochondrial membrane compositionin trout: responses of membrane enzymes and oxidative capacities

N. Martin • D. P. Bureau • Y. Marty •

E. Kraffe • H. Guderley

Received: 8 March 2012 / Revised: 16 August 2012 / Accepted: 8 September 2012

� Springer-Verlag Berlin Heidelberg 2012

Abstract To examine whether membrane fatty acid (FA)

composition has a greater impact upon specific components

of oxidative phosphorylation or on overall properties of

muscle mitochondria, rainbow trout (Oncorhynchus

mykiss) were fed two diets differing only in FA composi-

tion. Diet 1 was enriched in 18:1n-9 and 18:2n-6 while

Diet 2 was enriched in 22:6n-3. The FA composition of

mitochondrial phospholipids was strongly affected by diet.

22:6n-3 levels were twice as high (49 %) in mitochondrial

phospholipids of fish fed Diet 2 than in those fed Diet 1.

18:2n-6 content of the phospholipids also followed the

diets, whereas 18:1n-9 changed little. All n-6 FA, most

notably 22:5n-6, were significantly higher in fish fed Diet 1.

Nonetheless, total saturated FA, total monounsaturated FA

and total polyunsaturated FA in mitochondrial phospho-

lipids varied little. Despite a marked impact of diet on

specific FA levels in mitochondrial phospholipids, only

non-phosphorylating (state 4) rates were higher in fish fed

Diet 2. Phosphorylating rates (state 3), oxygen consump-

tion due to flux through the electron transport chain com-

plexes as well as the corresponding spectrophotometric

activities did not differ with diet. Body mass affected state

4 rates and cytochrome c oxidase and F0F1 ATPase

activities while complex I showed a diet-specific effect of

body mass. Only the minor FA that were affected by body

mass were correlated with functional properties. The reg-

ulated incorporation of dietary FA into phospholipids

seems to allow fish to maintain critical membrane functions

even when the lipid quality of their diets varies consider-

ably, as is likely in their natural environment.

Keywords Diet � Mitochondrial membranes �Phospholipids � Fatty acids � Membrane pacemaker �Oxidative phosphorylation � Principal component analysis

Introduction

Dietary lipid composition influences the fatty acid (FA)

composition of tissue phospholipids in a wide range of taxa

(Withers and Hulbert 1987; McKenzie et al. 1998; Hulbert

et al. 2005; Guderley et al. 2008; Lemieux et al. 2008;

Abbott et al. 2012). This presents a potential conflict as

membrane dynamics are sensitive to the FA composition of

phospholipids which can modify lipid–protein interactions

and protein function (Dowhan 1997; Phillips et al. 2009).

Various aspects of membrane FA composition are corre-

lated with rates of the membrane processes that are primary

determinants of basal metabolic rates (Hulbert and Else

1999, 2000, 2005). Regulation of membrane lipid charac-

teristics would seem essential for optimisation of energy

metabolism. Animals show considerable evidence for such

Communicated by G. Heldmaier.

N. Martin (&) � H. Guderley

Departement de Biologie, Universite Laval,

Quebec, QC G1K 7P4, Canada

e-mail: [email protected]

D. P. Bureau

UG/OMNR Fish Nutrition Research Laboratory,

Department of Animal and Poultry Science,

University of Guelph, Guelph, ON N1G 2W1, Canada

Y. Marty

Unite mixte CNRS 6521, Universite de Bretagne,

C.S. 93837, 29238 Brest cedex 3, France

E. Kraffe

LEMAR UMR CNRS 6539, Institut Universitaire Europeen

de la Mer, Place Copernic, Technopole Brest Iroise,

29280 Plouzane, France

123

J Comp Physiol B

DOI 10.1007/s00360-012-0712-5

Author's personal copy

regulation, ranging from tissue and organelle differences to

allometric changes in membrane lipid characteristics.

Furthermore, ectothermal animals regulate membrane lipid

characteristics when faced with changes in temperature or

salinity, presumably to maintain function in their season-

ally variable habitats (Hazel and Williams 1990). Although

it is clear that the FA composition of tissue phospholipids

is modified by diet, the impact of such changes on bio-

chemical and physiological processes is still a matter of

debate. Essentially, the question is whether animals allow

their membrane composition to follow their diet to such an

extent that crucial membrane functions are altered.

The difficulty in elucidating how dietary manipulation

of membrane FA composition affects physiological func-

tion is in part due to the variety of organisms, tissues and

cellular compartments studied, as each possesses its own

mechanisms regulating membrane composition, in part to

differing dietary formulations and in part to the range of

functions examined. In the following, we focus upon

mitochondria and their responses to dietary FA availability.

FA of mitochondrial membrane lipids are markedly influ-

enced by diet (Rohrbach 2009). The strongest evidence for

functional consequences of such modifications comes from

studies of mammals. Dietary FA composition modifies the

FA composition of phospholipids in rat heart and liver

mitochondria, but does not change oxidative capacities

(Astorg and Chevalier 1991; Lemieux et al. 2008) or

flux through electron transport chain (ETC) complexes

(Lemieux et al. 2008). On the other hand, heart mito-

chondria from rats fed an n-3-rich diet had reduced COX

activity and increased F0F1 ATPase activity compared to

those from rats fed an n-6-rich diet (Yamaoka et al. 1988).

After exposure to calcium, mitochondria enriched in n-3

PUFA had reduced complex I activity relative to control

mitochondria (Malis et al. 1990). Feeding rainbow trout a

diet rich in highly polyunsaturated fatty acids (PUFA)

increased the 22:6n-3 content in red muscle, enhanced

expression of carnitine palmitoyltransferase I mRNA but

did not change the functional properties of carnitine pal-

mitoyltransferase I (Morash et al. 2009). Manipulation of

dietary FA composition led to major remodelling of FA in

phospholipids of red muscle mitochondria from rainbow

trout, but only had minor effects on mitochondrial oxida-

tive capacities (Guderley et al. 2008). Overall, functional

impacts of diet-induced changes in FA composition seem

more apparent at the level of specific membrane-bound

enzymes, such as COX and F0F1 ATPase, than at the level

of organelle function (e.g. mitochondrial oxygen uptake).

This could be due to regulatory dynamics buffering chan-

ges in individual protein activities when they are integrated

into a larger system. It could also be that dietary effects at

higher levels of integration are subtle, requiring larger

sample sizes for their demonstration.

Given these considerations, we built upon our previous

study which showed a limited impact of dietary FA on

overall mitochondrial capacities (state 3, state 4) (Guderley

et al. 2008) by examining specific ETC components in

intact and disrupted mitochondria to evaluate whether

activities of enzymes in the mitochondrial membrane are

more sensitive to dietary manipulation than the overall

mitochondrial capacities. To this end, we fed juvenile

rainbow trout (Oncorhynchus mykiss) with two diets that

modified the FA composition of mitochondrial mem-

branes. We measured oxygen consumption due to flux

through ETC complexes as well as measuring maximal

activity of ETC complexes and F0F1 ATPase by spectro-

photometry. In the same preparations, we measured state 3

with pyruvate as substrate, state 4 and state 4 inhibited

with oligomycin (State 4ol). Concentrations of the adenine

nucleotide translocase (ANT) and cytochromes a, b, c and

c1 were quantified. In parallel, we characterised the FA

composition of mitochondrial phospholipids. All steps

were performed on sufficient animals to allow the appli-

cation of multivariate statistics to analyse how FA char-

acteristics responded to dietary FA and to identify

membrane FA characteristics correlated with functional

variability.

Materials and methods

Dietary composition

The experimental diets (Diet 1: enriched in 18:1n-9 and

18:2n-6, Diet 2: enriched in 22:6n-3) were isoproteic and

isoenergetic (460 g crude protein, 170 g lipid, and 20 mJ

digestible energy/kg) but used different lipid sources to

achieve the desired composition (Table 1). These diets met

known nutrient requirements of rainbow trout using highly

digestible ingredients. The 22:6n-3 was provided by drum-

dried algal (Schizochytrium sp.) meal, allowing us to avoid

the use of fisheries by-products. The diets were mixed

using a Hobart mixer (Hobart, Don Mills, ON, Canada) and

pellets of an appropriate size were made using a California

pellet mill (California Pellet Mill, Crawfordsville, IN,

USA). The pellets were dried under forced air at 22 �C for

24 h and then sieved. The diets were stored at -20 �C,

only the amount required for a week was kept at 4 �C.

Total SFA did not differ between diets with an approximate

value of 30 % in both (Table 2). MUFA differed between

diets, with 51 % in Diet 1 and 34 % in Diet 2. Only 18 %

of FA in Diet 1 were polyunsaturated in comparison to

32 % in Diet 2. The diets differed markedly in their % n-3,

% n-6, PUFA balance (defined as % n-3 of total PUFA) as

well as their double bond index (DBI). DBI was calculated

according to the following equation:

J Comp Physiol B

123


DBI ¼X

MUFA�þX

2UFA� 2þX

3UFA� 3

þX

4UFA � 4þX

5UFA � 5

þX

6UFA � 6

in which,P

MUFA is defined as the total of unsaturated

FA with one double bond andP

2 UFA as the total of

unsaturated FA with two double bonds, etc.

A few major FA accounted for the principal differences

in FA composition. 22:6n-3 content differed by about

25-fold, from less than 0.5 % (Diet 1) to 13 % (Diet 2) of

total FA. 18:2n-6 accounted for 18 % of FA in Diet 1 and

11 % in Diet 2. Similarly, 18:1n-9 accounted for 44 % of

total FA in Diet 1 and 30 % in Diet 2.

Fish holding

Experimental procedures were approved by our local ani-

mal ethics committee (CPAUL) and followed the guide-

lines of the Canadian Council on Animal Care (http://www.

ccac.ca). Juvenile rainbow trout (O. mykiss) were obtained

from a fish hatchery (Pisciculture de la Jacques Cartier,

Cap-Sante, QC, Canada) in late June 2008 at a water

temperature of 11 �C and transferred to the LARSA

(Laboratoire Regional des Sciences Aquatiques) at Uni-

versite Laval. During the first 2 weeks, fish were fed daily,

at a maintenance ration, with the diet (Corey Aquafeeds,

NB, Canada) used at the hatchery. The fish (initial mass

96 ± 12 g) were randomly distributed into 16 circular

tanks (60 l) with 8 replicates per diet and 10 fish per tank.

Throughout the experiment, photoperiod was maintained at

12 h light, 12 h dark and water temperature at 15 �C. Fish

were hand fed (3 times a day) to near satiety and weighed

every 28 days. After the 12th week, fish were transferred

into four tanks (1,000 l) with 2 replicates per diet and

reared for at least another week before mitochondrial

measurements started. Measurements were carried out over

Table 1 Composition of the diets (g/kg)

Ingredients Diet 1 Diet 2

Blood meal, whole, spray-dried 150 150

Casein, high N 250 250

Corn gluten meal 50 50

Soy protein concentrate 200 200

S-Type God Algae Flakesa – 160

Wheat middlings 150 60

Olive oil 70 70

Lard 100 30

Vitamin premixb 10 10

Vitamin E 1 1

Choline chloride 2 2

Mineral premixc 5 5

CaHPO4 10 10

Rovimixsaty-C 2 2

a S-Type Algae Flake from Advanced BioNutrition Corp (Columbia,

MD, USA)b Provides per kg of diet 2,500 IU of retinyl acetate, 2,000 IU of

cholecalciferol, 50 IU of 2DL-a-tocopherol-acetate, 1 mg of mena-

dione Na-bisulfate, 0.02 mg of cyanocobalamine, 50 mg of ascorbic

acid monophosphate, 0.14 mg of D-biotin, 1,000 mg of choline

chloride, 1.5 mg of folic acid, 4,500 mg of myo-inositol, 5 mg of

pyridoxine–HCl, 2 mg of riboflavine, 6 mg of thiamin–HClc Provides per kg of diet 1,200 mg of sodium chloride (39 % Na,

61 % Cl), 13 mg of ferrous sulphate, 32 mg of manganese sulphate,

60 mg of zinc sulphate, 7 mg of copper sulphate, 8 mg of potassium

iodide, 0.25 mg of selenium

Table 2 Fatty acid composition of diets

Fatty acid Diet 1 Diet 2

14:0 1.23 5.77

15:0 0.09 0.19

16:0 21.64 22.88

17:0 0.24 0.13

18:0 7.54 3.75

20:0 0.21 0.18

22:0 0.07 0.10

16:1n-9 0.28 0.16

16:1n-7 2.82 1.99

17:1n-8 0.23 0.19

18:1n-9 44.39 30.36

18:1n-7 2.41 1.45

20:1n-9 0.42 0.21

18:2n-6 17.60 10.62

20:2n-6 0.26 0.12

20:3n-6 0.07 0.18

20:4n-6 0.26 0.95

20:3n-3 0.04 0.09

20:4n-3 – 0.33

20:5n-3 0.02 0.58

22:4n-6 0.06 0.07

22:5n-6 0.01 4.88

22:5 n-3 0.03 0.13

22:6n-3 0.07 13.50

Total SFA 31.01 33.01

Total MUFA 50.56 34.42

Total PUFA 18.43 32.58

PUFA balance 0.91 48.04

Total (n-6) 18.26 16.82

Total (n-3) 0.17 15.59

(n-3)/(n-6) 0.01 0.93

DBI 86.69 170.88

Values are given in mol % of total fatty acids (FA). PUFA balance is

defined as % n-3 of total PUFA

DBI double bond index

J Comp Physiol B

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http://www.ccac.ca

http://www.ccac.ca

5 months, during which sampling alternated between the

dietary groups. Final anatomic characteristics were not

affected by diet (Table 4). As the trout grew considerably

over this period (the first trout weighed *300 g whereas

the trout sampled towards the end weighed *750 g), we

included fish mass as a covariate in our statistical analysis

(see below).

Tissue sampling and mitochondrial isolation

Fish were stunned by a blow on the head and rapidly killed

by severing the spinal cord. After measuring body mass

and length, liver and guts were removed and weighed. Red

muscle was used to isolate mitochondria. Muscle from both

sides of the animal was removed and red muscle was freed

of white muscle. All manipulations were carried on ice

except centrifugations, which were performed at 4 �C.

Mitochondria were isolated according to Kraffe et al.

(2007). This approach involved removal of the superficial

lipid layer after the first centrifugation (900g for 10 min) of

the homogenate. The supernatant from the first centrifu-

gation was centrifuged a second time at low-speed (900g),

to insure removal of heavy cellular debris and nuclei. The

remaining supernatant was then centrifuged at 9,000g for

10 min to obtain a mitochondrial pellet, from which MgCl2was removed by resuspension in isolation buffer free of

MgCl and re-centrifugation at 9,000g. The final mito-

chondrial pellet was re-suspended in a volume of reaction

buffer [140 mM KCl, 20 mM HEPES and 5 mM K2HPO4,

pH 7.3 with 0.5 % fatty acid free bovine serum albumin

(BSA)] corresponding to one-tenth of the mass of red

muscle used (i.e. 400 ll for 4 g of muscle). Immediately

after resuspension of the mitochondrial pellet, three ali-

quots were frozen (-80 �C) for subsequent spectrophoto-

metric assays of the activity of ETC components,

cytochrome concentrations and lipid extraction. An ali-

quot of the mitochondrial preparation was centrifuged

(9,000g for 10 min at room temperature) and the pellet was

re-suspended in reaction buffer without BSA and centri-

fuged again. The supernatant was discarded and the pellet

re-suspended, washed in reaction buffer and centrifuged

twice more to remove the BSA. The pellet was frozen

(-80 �C) in a volume of Millipore water corresponding to

the initial volume of the aliquot for subsequent determi-

nation of protein concentration (see below).

Polarographic measurement of mitochondrial oxidative

capacities and electron flux through ETC complexes

Oxygen consumption was measured with fresh mitochon-

drial preparations (final concentration of *0.25 mg protein

ml-1) using a temperature-controlled polarographic O2

monitoring system (Qubit System, Kingston, ON, Canada).

Temperature was maintained at 15 �C by a circulating

refrigerated water bath (Haake G8, Polyscience, Niles, IN,

USA). The oxygen probes were calibrated with air-satu-

rated reaction buffer and corrected for temperature and

atmospheric pressure. All components were dissolved in

reaction buffer, except oligomycin, rotenone, antimycin

and N,N,N0,N0-tetramethyl-p-phenylenediamine (TMPD)

that were dissolved in 95 % ethanol. All polarographic

rates were measured at least twice for all preparations.

For measurements of maximal oxidative capacities, the

Krebs cycle was sparked with addition of malate to a final

concentration of 0.37 mM. Pyruvate was provided as

the carbon substrate at final concentration of 3.45 mM.

Maximal state 3 rates were obtained after adding adenosine

diphosphate (ADP) to a final concentration of 0.92 mM.

State 4 rates were evaluated after depletion of ADP, once

oxygen uptake rates had stabilised. After measurement of

state 4 rates, oligomycin (1 mg ml-1 final concentration)

was added (State 4ol) to evaluate oxygen consumption in

the absence of oxidative phosphorylation (Estabrook

1967). The respiratory control ratio (RCR) was calculated

according to Estabrook (1967). Only preparations with

RCR values greater than 4 were conserved for further

analysis.

Oxygen consumption due to flux through the ETC

complexes was assessed on the day of mitochondrial iso-

lation following Martin et al. (2009). Oxygen consumption

due to flux through complexes I–IV was estimated from

rates of pyruvate ? malate oxidation (3.45 and 0.37 mM)

in the presence of 5 mM ADP and corrected for residual

rates after inhibition of complex I by rotenone (1 lg ml-1

final concentration). Preliminary experiments showed that

5 mM ADP was sufficient to maintain state 3 rates for the

time required for the complete series of measurements.

Subsequently, succinate (6 mM) was added to stimulate

flux through complexes II–IV. After steady rates were

reached, antimycin A (5 lg ml-1 final concentration) was

added to inhibit complex III. Finally, ascorbate (8 mM)

and TMPD (0.8 mM) were added to stimulate flux through

complex IV (COX). These rates were corrected for auto-

oxidation of TMPD in the presence of ascorbate, after

inhibition of COX by potassium azide (70 lM). Pre-

liminary experiments established optimal substrate and

inhibitor concentrations for these measurements from trout

mitochondria.

Spectrophotometric assays

These determinations used mitochondrial samples that had

been submitted to two cycles of freezing and thawing.

Assays were run at 15 �C using a UV/Vis spectropho-

tometer (Beckman DU 640, Beckman Instruments,

J Comp Physiol B

123


Fullerton, CA, USA) with a temperature-controlled cell

holder and circulating refrigerated water bath (Haake G8).

All assays were run at least in triplicate. Preliminary

experiments established optimal substrate and inhibitor

concentrations for these enzymes from trout mitochondria.

The assay for complex I (NADH-CoQ reductase) was

based on Paradies et al. (2002). The reaction mixture

contained 50 mM phosphate buffer, pH 7.2, 0.1 % fatty

acid free BSA, 3 mM KCN, 0.64 lg ml-1 antimycin A,

150 lM decylubiquinone (DUB, dissolved in ethanol) and

60 lM NADH. Initially, mitochondrial extract (*20 lg

protein ml-1), NADH, antimycin A and KCN were incu-

bated together for 240 s before the addition of DUB. After

letting the assay mixture stabilise for 60 s, the decrease in

optical density of NADH was followed for 240 s at

340 nm. The inhibition due to the addition of rotenone

(1 lg ml-1 final concentration) indicated the activity of

complex I. Activities were calculated using an extinction

coefficient of 6.22 mM-1 cm-1 and are expressed as lmol

of NADH oxidised per minute.

COX activity was measured according to Bouchard and

Guderley (2003), except that mitochondrial suspensions

were diluted in phosphate buffer without Triton-X (45 mM

KH2PO4 and 30 mM K2HPO4, pH 6.8). An initial cyto-

chrome c concentration of 100 lm was used. The mito-

chondrial extract was diluted to *5 lg protein ml-1. The

cytochrome c solution was reduced by the addition of a few

grains of sodium hydrosulfite. Bubbling with air, at least

30 min, eliminated excess hydrosulfite. The ratio of the

optical density at 550 and 565 nm was used to insure

that cytochrome c was sufficiently reduced. Activities

were calculated using an extinction coefficient of

19.1 mM-1 cm-1 and are expressed as lmol of cyto-

chrome c oxidised per minute (first order reaction).

F0F1 ATPase activity was measured by coupling the

production of ADP to the oxidation of NADH via the

pyruvate kinase and lactate dehydrogenase reactions

(Zheng and Ramirez 1999; Itoi et al. 2004). The reaction

mixture contained 5 mM KCN, 0.3 mM NADH, 4 mM

MgATP, 1 mM phosphoenolpyruvate, 2 mM MgCl2,

100 mM Tris–HCl at pH 7.5 as well as 10 Units of pyru-

vate kinase and 38.5 Units of lactate dehydrogenase per ml

assay medium. Initially, the optical density was followed

for 400 s to insure that the contaminating ADP in

the adenosine triphosphate (ATP) was consumed.

Then *20 lg protein ml-1 of mitochondrial extract were

added and the decrease in NADH absorbance at 340 nm

was followed for 400 s. Oxidation of one molecule of

NADH is coupled to the consumption of one molecule of

ATP by the F0F1 ATPase. Oligomycin (10 lg ml-1 final

concentration) was added to quantify the NADH oxidation

linked to ATPase activity of the F0F1 ATP synthase.

Oligomycin was dissolved in ethanol.

Adenine nucleotide translocase, cytochrome

and protein concentrations

The concentration of adenine nucleotide translocase (ANT)

was measured in fresh mitochondrial preparations by

titration with its noncompetitive and irreversible inhibitor,

carboxyatractyloside (CAT) (Guderley et al. 2005). State 3

respiration was gradually inhibited with repeated additions

of CAT and the inhibition was considered complete when

addition of CAT had no further effect on oxygen uptake.

Subsequent addition of 2 lM CCCP (carbonyl cyanide

m-chlorophenylhydrazone) confirmed that the ETC was

still functional (Brand et al. 2005). The titration was plotted

(state 3 rates vs. CAT concentration added), and the

concentration of ANT was determined from the CAT

concentration at which the line of steepest slope intersected

the fully inhibited state (Brown et al. 2007). The quantity

of CAT at this point was equal to the amount of ANT in the

mitochondrial suspension.

Cytochromes a, b, c, and c1 were quantified by difference

spectra according to Sherratt et al. (1988). Mitochondrial

preparations were disrupted by freeze–thawing and a 200 ll

aliquot (*13 mg protein ml-1) was incubated with rote-

none (1 lg mg-1 protein) in a final volume of 1 ml of

isolation buffer (oxidised cuvette) to fully oxidise the ETC

cytochromes (Sherratt et al. 1988). Rotenone was dissolved

in ethanol. Subsequently, the ETC was reduced by the

addition of 10 mM succinate, 40 lM ADP and 1 mM KCN.

The oxidised–reduced difference spectrum was recorded to

reveal absorption peaks of reduced respiratory pigments

using a UV/Vis spectrophotometer (Beckman DU 640) at

room temperature. We used the simultaneous equations

described by Schneider et al. (1980) to calculate the con-

centrations of individual cytochromes.

Protein concentration in mitochondrial preparations was

determined by the bicinchoninic acid method (Smith et al.

1985) using free fatty acid BSA as the standard.

Membrane lipid analysis

Lipids were extracted from a 50-ll aliquot of mitochon-

drial preparation according to Folch et al. (1957). The

extract, including phospholipids from the inner and outer

mitochondrial membranes, was stored at -80 �C under

nitrogen atmosphere after adding 0.01 w/v of butylated

hydroxytoluene (BHT). An aliquot of the lipid extract

was evaporated to dryness and lipids were recovered with

three washings of 500 ll of CHCl3/methanol (98:2, v/v)

and deposited at the top of a silica gel micro-column

(30 mm 9 5 mm inner diameter, packed with Kieselgel 60

(70–230 mesh, Merck KGaA Corporate Procurement,

Darmstadt, HE, Germany) previously heated at 450 �C and

deactivated with 6 wt% H2O (Marty et al. 1992). Neutral

J Comp Physiol B

123


lipids were eluted with 10 ml of CHCl3/methanol (98:2,

v/v). The polar lipid fraction was recovered with 20 ml of

methanol and stored at -20 �C for further analysis of FA

composition by gas chromatography (Varian CP 8400,

Varian, Middelburg, ZE, The Netherlands). The polar lipid

fraction (phospholipids) was transesterified for 10 min at

100 �C after adding 800 ll of MeOH/BF3, 0.01 % w/w

BHT as an antioxidant and C23:0 FA as the internal

standard. After cooling and adding 800 ll of hexane, the

organic phase containing fatty acid methyl esters was

washed three times with 1.5 ml of hexane-saturated water.

Fatty acid methyl esters obtained were identified and

quantified using both polar (CPWAX 52 CB, Varian,

Middelburg, ZE, The Netherlands; 50 m 9 0.25 mm i.d.;

0.2 lm thickness) and nonpolar (CP-sil 8 B, Varian;

25 m 9 0.25 mm i.d; 0.25 lm thickness) capillary col-

umns. FA were expressed as molar percentage (mol %) of

total FA content.

Statistical analysis

As both diet and body mass influenced mitochondrial

capacities (Table 3), we used analysis of covariance with

mass as a covariate, assuming a linear regression model

(Milliken and Johnson 2001). If body mass did not affect a

parameter, we used ANOVA to assess whether it was

affected by diet. When body mass affected a parameter and

the mass dependence did not differ with diet, we applied a

parallel lines model (ANCOVA) with body mass as

covariate and diet as the factor. If the effect of body mass

on a given functional parameter changed with diet, we

compared the parameter for the two dietary groups at 400,

500 and 600 g body mass, using the LS means estimated by

the regression model.

With the aim of determining which FA best explained

the variability of the functional parameters, all FA from the

mitochondrial phospholipids were included in a principal

component analysis (PCA). PCA distils a set of potentially

correlated variables into a set of linearly uncorrelated

principal components, in our case establishing the influence

of the different FA on the principal components. PCA

allows the examination of the variability in mitochondrial

FA as a whole, rather than by inspection of individual

changes. After carrying out the PCA, we examined the

relationships between individual scores for these principal

components and functional parameters measured. We

applied a Bonferroni correction (Rice 1989) to adjust the

significance levels of the tests for the number of compar-

isons made. Finally, we examined whether the FA linked to

the principal components that were significantly correlated

with functional parameters were themselves linked with

these functional parameters. The significance level for

these tests was adjusted as above. Statistical analysis was

performed using SAS 9.2 software.

Results

Fish status

The externally measured variables of the animals used did

not differ between dietary groups (Table 4). There were no

Table 3 F statistics for analysis of parameters affected by body mass or by an interaction between diet and body mass

Diet Body mass Body mass 9 diet

Cytochrome c1 F2,51 = 0.02 F2,51 = 7.24** F2,51 = 3.87**

Total phospholipids F2,55 = 0.18 F2,55 = 6.14* F2,55 = 3.90*

State 4 F2,55 = 7.60** F2,55 = 7.45** F2,55 = 6.23**

State 4 oligo F2,51 = 0.21 F2,51 = 8.58** F2,51 = 4.84**

Complex I F2,54 = 3.07** F2,54 = 9.20** F2,54 = 7.39**

COX F2,46 = \ 0.001 F2,46 = 19.4*** F2,46 = 9.35***

F0F1 ATPase F2,42 = \ 0.001 F2,42 = 39.9*** F2,42 = 23.5***

24:1n-11 F2,55 = 1.40 F2,55 = 16.2*** F2,55 = 10.2***

24:1n-9 F2,55 = 0.68 F2,55 = 10.2*** F2,55 = 9.61***

24:1n-7 F2,55 = 5.34* F2,55 = 6.11* F2,55 = 7.22**

22:5n-6 F2,55 = 64.8*** F2,55 = 7.53** F2,55 = 42.6***

22:5n-3 F2,55 = 36.1*** F2,55 = 21.8*** F2,55 = 24.2***

n = 27 for fish fed Diet 1 and n = 32 for fish fed Diet 2. Cytochrome c1 as well as total phospholipids are expressed as nmol mg-1 protein.

Cytochrome c1 and Complex I activity showed an interaction between diet and body mass. The impact of diet was assessed via LS mean

comparisons at 400, 500 and 600 g of body mass. For all the remaining parameters in this table, we examined the impact of dietary treatment

with an ANCOVA, using diet as the factor and body mass as the covariate. All other measured parameters that are not shown in this table, were

analysed via analysis of variance (ANOVA) with diet as the factor. Data are given as F statistics for the factor diet, the covariate body mass and

their interaction. * p B 0.05, ** p B 0.01, *** p B 0.001. We show the Fx,y statistics as x: number of treatments and y: degrees of freedom

J Comp Physiol B

123


significant differences in the mass of liver, heart and

digestive tract or the hepatosomatic index. No gender

effects were observed as all of the individuals were female

with the exception of one male.

Fatty acid composition of mitochondrial phospholipids

The relative abundance of virtually all major FA ([1 %) as

well as some minor FA (\1 %) in the total mitochondrial

phospholipids was affected by diet (Table 5). In most

cases, the effect was highly significant (p \ 0.0001). Many

characteristics followed the differences in the diets;

including % n-3, % n-9, n-3/n-6, and the following FA:

18:0, 18:1n-9, 18:2n-6, 20:2n-6 and 22:6n-3. However,

some FA in mitochondrial phospholipids changed in the

opposite direction to the dietary FA profile, including

20:3n-6, 20:4n-6, 20:5n-3, 22:5n-3 and 22:5n-6. Certain

properties of mitochondrial phospholipids, in particular

22:4n-6 and % n-6, differed significantly between dietary

treatments, even though they were similar in the diets.

The SFA of mitochondrial phospholipids were domi-

nated by 16:0 while 18:1n-9, 18:2n-6 and 22:6n-3 were the

main unsaturated FA (Table 5). The FA composition of

mitochondrial phospholipids isolated from red muscle

following the two dietary treatments, showed similar levels

of 16:0; *14.5 % in both groups. The level of 22:6n-3 in

the phospholipids was highly affected by diet, being 24 %

in fish fed Diet 1 and 49 % in fish fed Diet 2. 18:2n-6

showed the opposite pattern and was higher (9 %) in

mitochondrial phospholipids of fish fed Diet 1 than in those

fed Diet 2 (4 %). Although its levels differed markedly

between diets (44 % in Diet 1 vs. 30 % in Diet 2), 18:1n-9

differed only by 3 % in mitochondrial phospholipids. This

difference was significant. All the n-6 FA quantified

(18:2n-6, 20:2n-6, 20:3n-6, 20:4n-6, 22:5n-6 and 24:4n-6)

were significantly higher in phospholipids of fish fed Diet

1. Many of the overall differences between the diets (total

PUFA, total MUFA and PUFA balance) were not reflected

in the acyl composition of mitochondrial membranes. Total

PUFA differed significantly but was 64 % in fish fed Diet 1

and 68 % in fish fed Diet 2 (dietary contents 18 vs. 33 %).

PUFA balance was 42 % in fish fed Diet 1 and 75 % in fish

fed Diet 2 (dietary values 0.91 vs. 48 %). Total MUFA also

Table 4 Anatomic characteristics of trout used for mitochondrial

isolation

Diet 1 Diet 2

Initial body mass (g) 100.32 ± 2.30 100.16 ± 3.20

Initial length (cm) 20.30 ± 0.40 20.10 ± 0.52

Final body mass (FM; g) 474.30 ± 22.06 475.30 ± 22.03

Final length (cm) 31.50 ± 0.52 31.72 ± 0.4

Final eviscerated mass (g) 396.30 ± 16.00 404.22 ± 16.00

Liver mass (LM; g) 5.30 ± 0.30 5.22 ± 0.31

Digestive system (g) 43.10 ± 2.60 41.13 ± 2.12

Heart (g) 0.48 ± 0.02 0.49 ± 0.02

Red muscle (g) 7.34 ± 0.31 7.60 ± 0.26

Fulton Index (FM/L3) 9 100 1.38 ± 0.05 1.35 ± 0.02

Hepatosomatic index

(LM/FM) 9 100

1.11 ± 0.03 1.09 ± 0.04

Data are shown as mean ± SEM, n = 27 for the group fed Diet 1 and

n = 32 for the group fed Diet 2. The anatomic characteristics did not

differ between dietary treatments (ANOVA p [ 0.5)

Table 5 Fatty acid composition of the total phospholipids isolated

from red muscle mitochondria of trout fed different diets

Fatty acid Diet 1 Diet 2

16:0a 14.51 ± 0.42 14.72 ± 0.40

18:0a* 4.71 ± 0.11 4.51 ± 0.04

18:1n-9a*** 12.91 ± 0.30 9.41 ± 0.23

18:1n-7a 1.73 ± 0.20 1.62 ± 0.06

18:2n-6a*** 9.10 ± 0.39 3.72 ± 0.31

20:2n-6a*** 1.30 ± 0.06 0.50 ± 0.03

20:3n-6a*** 4.00 ± 0.27 0.51 ± 0.14

20:4n-6a*** 6.52 ± 0.29 2.42 ± 0.21

20:5n-3a* 1.12 ± 0.08 0.91 ± 0.07

22:4n-6a*** 1.63 ± 0.08 0.82 ± 0.07

22:5n-6b*** 14.22 ± 0.60 8.93 ± 0.35

22:5n-3b*** 1.13 ± 0.04 0.80 ± 0.04

22:6n-3a*** 23.72 ± 1.22 48.93 ± 1.30

Others 3.51 ± 0.27 2.32 ± 0.15

Total SFAa 19.93 ± 0.62 19.91 ± 0.42

Total MUFAa*** 16.54 ± 0.33 12.32 ± 0.31

Total PUFAa*** 63.53 ± 0.64 67.84 ± 0.52

PUFA balancea*** 41.83 ± 2.12 75.12 ± 1.51

Total (n-9)a*** 14.13 ± 0.33 10.11 ± 0.26

Total (n-7)a 2.23 ± 0.22 1.92 ± 0.12

Total (n-6)a*** 36.84 ± 1.33 16.91 ± 0.93

Total (n-3)a*** 26.75 ± 1.30 50.21 ± 1.23

(n-3)/(n-6)a*** 0.84 ± 0.12 3.31 ± 0.14

DBIa*** 309.00 ± 4.00 383.00 ± 5.00

Values are expressed as mol % and mean ± SEM, n = 27 for the

group fed Diet 1 and n = 32 for the group fed Diet 2. Of the 25 FA

that were detectable, the following FA were below 1.0 % and com-

piled under the name others: 14:0, 15:0, 17:0, 20:0, 22:0, 24:0, 16:1n-

9, 16:1n-7, 20:1n-9, 20:1n-7, 24:1n-11, 24:1n-9, 24:1n-7, 20:3n-3,

20:4n-3, 24:6n-3 and 24:4n-6

Bold FA were affected by diet. PUFA balance is defined as % n-3 of

total PUFA

DBI double bond index

Asterisk indicate values that differ with dietary treatment * p B 0.05,

** p B 0.01, *** p B 0.0001)

Letters indicate the type of analysis used, see ‘‘Materials and meth-

ods’’, a ANOVA, b ANCOVA with a body mass effect (p B 0.05)

J Comp Physiol B

123


showed a limited albeit significant difference, being 17 %

in fish fed Diet 1 and 13 % in fish fed Diet 2 (dietary values

51 vs. 34 %). Total SFA were maintained between dietary

treatments. DBI was 20 % higher in fish fed Diet 2 than in

those fed Diet 1 (Table 5). The concentration of phos-

pholipids was *600 nmol total phospholipids mg-1 pro-

tein and did not differ with dietary treatment but decreased

with body mass (R2: 0.17, Table 6).

A few FA in mitochondrial phospholipids changed with

body mass, in a similar fashion in the two groups (Table 3;

Fig. 1). The 24:1 series FA were negatively correlated with

body mass (24:1n-11, R2 0.25, p \ 0.0001; 24:1n-9, R2

0.25, p \ 0.0001; 24:1n-7, R2 0.13, p 0.0052). 22:5n-6

increased with body mass (R2 0.15, p 0.0032), whereas

22:5n-3 decreased (R2 0.12, p 0.0081). Doubling body

mass of 22:5n-6 and 22:5n-3 led the proportions of these

FA to change by approximately 30 %.

Protein composition of mitochondria

The concentrations of cytochromes a, b, c, and c1 and ANT

(expressed as nmol mg-1 protein) in isolated mitochondria

were not significantly affected by dietary treatment

(Table 6). Cytochrome c1 showed an impact of body mass

and an interaction between diet and body mass, but no

significant impact of diet on cytochrome c1 levels was

found at 400, 500 or 600 g. We examined our functional

data using these different components as denominators and

found that, in general, the same conclusions were obtained

with all denominators. Thus, we present the functional data

using the denominator most frequently used in the mito-

chondrial literature, mg mitochondrial protein (mg-1

protein).

Oxidative capacities and oxygen consumption

due to flux through respiratory chain complexes

assessed by polarography

Dietary treatment did not affect rates of pyruvate ? malate

oxidation (state 2) nor maximal state 3 rates (Fig. 2). In

contrast, dietary treatment significantly affected state 4

rates. Rates were approximately 30 % higher in fish fed

Diet 2 (Fig. 2). State 4 rates were positively correlated with

body mass (R2: 0.07, p: 0.0384, Fig. 5). After inhibition by

oligomycin, oxygen uptake did not differ with diet. RCR

was significantly higher for mitochondria from fish fed Diet

1 (6.19 ± 0.33 vs. 5.15 ± 0.29, p \ 0.05).

Oxygen uptake due to flux through respiratory chain

complexes was not affected by dietary treatment (Fig. 3).

Rates of oxygen consumption linked to flux through COX

were approximately 10-fold for those through complexes

II–IV in fish fed both diets. Diet did not affect the relative

rates of complexes I–IV and complexes II–IV.

Maximal activities of the respiratory chain complexes

assessed by spectrophotometry

Spectrophotometric assays measured the activity of respi-

ratory chain complexes in disrupted mitochondria provided

with maximal levels of exogenous substrates. Similar to

when these parameters were measured by polarography in

intact mitochondria, they showed no impact of diet

(Fig. 4). The relative activities of the complexes also did

not differ with diet.

COX activity increased with body mass (R2 0.30,

p \ 0.0001), whereas F0F1 ATPase activity decreased

(R2 0.53, p \ 0.0001; Fig. 5). For COX and F0F1 ATPase,

the mass effect in the dietary groups was parallel, with a

doubling of body mass leading to an approximately 50 %

change in activity. The impact of body mass on complex I

was positive only for fish fed Diet 1 (R2 0.33, p 0.0384;

Fig. 5). At 600 g body mass, complex I activity was 20 %

higher in fish fed Diet 1 than in fish fed Diet 2, but this

conclusion did not hold at 400 and 500 g.

Principal components analysis

Dietary modulation of FA in total mitochondrial phos-

pholipids was well demonstrated by PCA. About 60 % of

the variability in FA composition was explained by the first

three principal components (Fig. 6). In our analysis of

these components, we chose to interpret only major

descriptors (loadings [±0.20). Without exception, indi-

vidual FA were major descriptors of only one principal

component. Principal component 1 accounted for 35 % of

the variability and was mainly explained by the most

Table 6 Concentrations of cytochromes, adenine nucleotide trans-

locase (ANT) and phospholipids in mitochondria isolated from red

muscle of trout fed different diets

Diet 1 Diet 2

Cytochrome aa 0.67 ± 0.02 0.64 ± 0.02

Cytochrome ba 0.07 ± 0.01 0.06 ± 0.01

Cytochrome c1 (400)c 0.19 ± 0.01 0.17 ± 0.01

Cytochrome c1 (500)c 0.16 ± 0.01 0.16 ± 0.01

Cytochrome c1 (600)c 0.12 ± 0.01 0.16 ± 0.02

Cytochrome ca 0.14 ± 0.01 0.14 ± 0.01

ANTa 8.07 ± 0.40 8.21 ± 0.37

Phospholipidsb 640.2 ± 41.1 580.1 ± 30.2

Values are expressed as nmol mg-1 mitochondrial protein and are

given as mean ± SEM, n = 27 for trout fed the Diet 1 and n = 32 for

trout fed the Diet 2. None of these components changed with dietary

treatment (a ANOVA; p [ 0.1, b ANCOVA; p [ 0.1 c LS means

comparison at 400, 500 and 600 g of body mass, p [ 0.1)

J Comp Physiol B

123


Body mass (g)

Fat

ty a

cid

(% m

ol)

0

0.1

0.2

0.3

0.4

0.5

0.6

200 400 600 800

B

0

5

10

15

20

25

200 400 600 800

C

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

200 400 600 800

D

0.00

0.05

0.10

0.15

0.20

200 400 600 800

A

Fig. 1 Levels of 24:1n-11 (a), 24:1n-9 (b), 22:5n-6 (c) and 22:5n-3

(d) relative to body mass of fish. Linear regression equations are:

a y = -0.0002x ? 0.14, n = 58, R2 = 0.25; b y = 0.00004x ? 0.53,

n = 58, R2 = 0.25; c y = 0.0123x ? 5.29, n = 58, R2 = 0.15;

d y = -0.0008x ? 1.31, n = 57, R2 = 0.12. Filled circles corre-

spond to fish fed Diet 1 while empty circles represent fish fed Diet 2

0

20

40

60

80

100

120

140

160

State 2 State 3 State 4

na

no

ato

m O

min

-1 m

g-1

pro

tein Diet 1

Diet 2

**

State 4Ol

Fig. 2 Maximal rates of pyruvate oxidation (State 2), oxidative

capacities (State 3), non-phosphorylating (State 4) respiration and

oligomycin-inhibited State 4 (State 4ol) of mitochondria isolated from

red muscle of rainbow trout fed two different diets. Values are shown

as mean ± SEM, n = 27 for the group fed Diet 1 and n = 32 for

group fed Diet 2. Assay temperature was 15 �C. State 4 differed

between treatment, ANCOVA (p \ 0.01)

0

50

100

150

200

250

300

Flux I-IV Flux II-IV Flux COX

nano

ato

m O

min

-1 m

g-1

prot

ein

Diet 1

Diet 2

Fig. 3 Oxygen uptake due to the flux through complexes I–IV,

complexes II–IV and cytochrome c oxidase (COX) of isolated

mitochondria from red muscle of rainbow trout fed different diets. Assay

temperature was 15 �C. Data are shown as mean ± SEM, n = 27 for the

group fed Diet 1 and n = 32 for group fed Diet 2. Maximal flux through

complexes of the ETC did not differ statistically between groups

J Comp Physiol B

123


abundant FA, including: 18:1n-9, 20:1n-9, 18:2n-6, 20:2n-6,

20:3n-6, 20:4n-6, 20:4n-3, 22:4n-6, 22:5n-6 as well as

22:5n-3. All these FA showed similar positive loadings on

the first component, while 22:6n-3 (the most abundant FA

for both dietary groups) was the only FA with a negative

loading. This first PCA axis showed graphically that trout

mitochondria tend to replace 22:6n-3 with n-6 FA when

22:6n-3 is not available in the diet. The second principal

component was mainly influenced by SFA and accounted

for 14 % of the variability. FA influencing this compo-

nent were 15:0, 17:0, 18:0, 20:0 as well as minor FA such

as 16:1n-9 and 18:1n-7. Loadings for all these FA were

positive, with 16:1n-9 showing the highest loading.

Except for 14:0 and 18:0, FA that influenced the second

principal component were not altered by diet. The third

component explained 9 % of the variability and was

dominated by the 24 carbon chain FA (24:1n-7, 24:1n-9,

24:1n-11), 20:5n-3 and 22:5n-3. Body mass affected the

levels of these FA (with the exception of 20:5n-3)

(Table 3; Fig. 1). All the major descriptors of the third

0

200

400

600

800

1000

1200

CX I (400g) CX I (500g) CX I (600g) COX

nmol

of s

ubst

rate

s m

in-1

mg

-1pr

otei

n

Diet 1

Diet 2

**

F0F1 ATPase

Fig. 4 Spectrophotometric activity of ETC complexes of isolated

mitochondria from red muscle of rainbow trout fed different diets.

Assay temperature was 15 �C. Data are shown as mean ± SEM,

n = 27 for the group fed Diet 1 and n = 32 for the group fed Diet 2.

Asterisk indicates that values differ, p \ 0.01. An interaction between

diet and body mass affected complex I (CX I), complex I activity was

compared at 400, 500 and 600 g with Ls mean comparisons

Body mass (g)

0

100

200

300

400

500

600

200 400 600 800

A

0

500

1000

1500

2000

2500

200 400 600 800

B

0

50

100

150

200

250

200 400 600 800

C

0

10

20

30

40

50

60

200 400 600 800

D

Enz

ymat

ic a

ctiv

ity(U

mg

-1pr

otei

n)

Enz

ymat

ic a

ctiv

ity(U

mg

-1pr

otei

n)

Oxy

gen

cons

umpt

ion

(nat

om O

min

-1m

g-1

prot

ein)

Enz

ymat

ic a

ctiv

ity(U

mg

-1pr

otei

n)

Fig. 5 Activities of F0F1 ATPase (a), cytochrome c oxidase (COX—

b), complex I (c) and non-phosphorylating rates (State 4—d) relative

to body mass of fish (U mg-1 protein for enzymes and natom

O min-1 mg-1 protein for State 4 rates). Linear regression equations:

a y = -0.62x ? 584, n = 45, R2 = 0.53; b y = 1.65x ? 115,

n = 49, R2 = 0.30; c y = 0.20x ? 38, n = 27, R2 = 0.33 for Diet 1

and y = 0.04x ? 100, n = 32, R2 = 0.03 for Diet 2; d y = 0.02x ?

13, n = 57, R2 = 0.07. The effect of body mass was equivalent in the

two dietary groups for F0F1 ATPase, COX and State 4 rates while

Complex I activity showed a significant interaction between diet and

body mass. Filled circles correspond to fish fed Diet 1 while emptycircles represent fish fed Diet 2

J Comp Physiol B

123


0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45

14:0

15:0

16:0

17:0

18:0

20:0

22:0

24:0

16:1n-9

16:1n-7

18:1n-7

20:1n-9

20:3n-3

20:5n-3

Principal Component 2

-0.35 -0.30 -0.25 -0.20 -0.15 -0.10 -0.05 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35

14:0

24:0

18:1n-9

20:1n-9

24:1n-7

18:2n-6

20:2n-6

20:3n-6

20:4n-6

20:4n-3

20:5n-3

22:4n-6

22:5n-6

22:5n-3

22:6n-3

24:4n-6


-0.25 -0.20 -0.15 -0.10 -0.05 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0.50 0.55

14:0

15:0

16:0

17:0

18:0

22:0

24:0

16:1n-7

24:1n-11

24:1n-9

24:1n-7

20:3n-3

20:5n-3

22:4n-6

22:5n-6

22:5n-3


Fig. 6 Weight of the original

descriptors (specific fatty acid),

for the first three principal

components, from which

individual scores were

computed. Black bars indicate

variable for which loadings

were ± 0.20 while gray barsindicate loadings under this

threshold. Principal component

1 explains 35 % of the variance

while principal component 2

and 3 explain 14.8 and 9.2 %,

respectively. In order to reduce

complexity of the figure, only

FA with loading ± 0.1 were

presented

J Comp Physiol B

123


component showed positive loadings. 24:1n-9 and 24:1n-

11 had the strongest loadings.

Links between functional properties of trout muscle

mitochondria and the FA composition of their

phospholipids

After demonstrating the impact of diet upon functional and

structural properties of muscle mitochondria, our next step

was to evaluate links between these properties. General

descriptors of lipid structure such as % n-3, % n-6, n-3/n-6,

total MUFA, total PUFA and PUFA balance were not cor-

related with functional parameters. We next examined

whether individual values of the functional parameters

modified by diet or body mass were correlated with indi-

vidual scores for the principal components obtained from

the PCA of FA composition of total phospholipids. The

concentrations of various mitochondrial proteins (cyto-

chrome a, b, c, c1, ANT) were not correlated with the scores

of the principal components. Functional parameters were

only significantly correlated with scores of principal com-

ponent 3. These parameters included state 3, state 4ol as well

as spectrophotometric activity of COX and F0F1 ATPase.

We then examined whether the main descriptors (specific

FA) of component 3 were correlated with these parameters.

F0F1 ATPase was positively correlated with 24:1n-11

(R2: 0.42) and 24:1n-9 (R2 0.33; Table 7), whereas state 3,

COX and state 4ol were negatively correlated with 24:1n-9

(R2 0.15, 0.26 and 0.20, respectively; Table 7). COX was

also negatively correlated with 24:1n-7 (R2: 0.21). None of

the 24:1 FA were abundant in mitochondrial phospholipids.

Neither 24:1n-11 nor 24:1n-9 was affected by diet, how-

ever, both decreased with fish mass (Fig. 1). Levels of

24:1n-11 were *0.06 %, those of 24:1n-9 were *0.03 %.

Levels of 24:1n-7 were lower in fish fed Diet 1

(0.03 ± 0.006 % vs. 0.05 ± 0.006 %, p \ 0.05) and also

decreased with body mass (R2: 0.13).

Discussion

Dietary treatment dramatically changed the FA composi-

tion of total phospholipids of muscle mitochondria from

rainbow trout, but had little impact upon functional char-

acteristics, including mitochondrial respiratory capacities

(state 2, state 3, state 4ol), oxygen uptake due to flux

through ETC as well as maximal activities of ETC com-

plexes and F0F1 ATPase. Clearly the activities of ETC

components and the F0F1 ATPase were as insensitive

to dietary treatment as maximal oxidative capacities.

Diet also left the concentrations and relative levels of ANT,

cytochromes a, b, c, c1 and total phospholipid content

(expressed relative to mitochondrial protein content)

unchanged. The strong modifications of FA of total mito-

chondrial phospholipids agree with previous findings of

dietary effects on phospholipid composition in fish (Ushio

et al. 1997; Robin et al. 2003; Guderley et al. 2008), rats

(Yamaoka et al. 1988; Croset et al. 1989; Lemieux et al.

2008; Abbott et al. 2010) and birds (McCue et al. 2009).

Even though our literature review suggested that specific

membrane processes (e.g. COX and F0F1 ATPase) would

be more sensitive to membrane FA composition than

overall mitochondrial properties, our targeted comparison

of these levels of organisation using a large sample size did

not support this hypothesis.

Integration of dietary FA into mitochondrial phospho-

lipids shows considerable evidence of regulation. Mem-

brane FA composition did not simply reflect the diets, as

several n-3 and n-6 FA changed in the opposite direction to

the diets and some FA remained constant despite differing

between diets. For example, whereas levels of a major FA,

18:1n-9, were 44 % in Diet 1 and 30 % in Diet 2, mito-

chondrial phospholipids from trout fed these diets differed

little, being 12.9 and 9.4 %, respectively. Similarly, dietary

differences in 18:1 content are not reflected in mitochon-

drial phospholipids from trout muscle and liver (Guderley

Table 7 Correlations between

fatty acid proportions and

functional parameters of red

muscle mitochondria of trout

fed different diets

Correlations were interpreted

using the level of significance

assessed by the Bonferroni

correction (p \ 0.004)

Functional parameter Fatty acid p value R2 Direction

State 3 24:1n-9 0.003 0.14 ;

24:1n-11 0.057 0.06 ;

State 4Ol 15:0 0.064 0.06 :

24:1n-7 0.062 0.06 ;

24:1n-9 0.001 0.20 ;

COX (spectro) 24:1n-7 0.001 0.21 ;

24:1n-9 0.0002 0.26 ;

22:5n-3 0.025 0.10 ;

F0F1 ATPase 15:0 0.033 0.10 ;

24:1n-9 \ 0.0001 0.33 :

24:1n-11 \ 0.0001 0.42 :

22:5n-3 0.049 0.08 :

J Comp Physiol B

123


et al. 2008; Morash et al. 2009) or rat heart (Lemieux et al.

2008). Regulation by biosynthesis would explain the fact

that the FA within the n-6 elongation pathway were sys-

tematically higher in fish fed Diet 1, even though 18:2n-6

was the only n-6 FA that differed between diets. Perhaps

the most extreme case was that of 22:5:n-6 which was

accumulated by fish fed Diet 1, despite its virtual absence

from this diet. Such responses of the n-6 PUFA require the

activity of the D6 and D5 desaturases to lead to accumu-

lation of products of the elongation chain. A similar pat-

tern, in which levels of n-6 FA (and again 22:5n-6) were

higher in mitochondrial membranes from trout fed a more

saturated diet, led to maintenance of total PUFA in mito-

chondrial phospholipids from trout red muscle (Guderley

et al. 2008). We note that n-6 FA of phospholipids from

liver mitochondria were also enhanced in trout fed a diet

containing traces of 22:6n-3 (0.38 %) (Morash et al. 2009),

although, unfortunately, 22:5n-6 levels were not reported.

Thus, trout fed a diet that does not allow the animal to form

or directly obtain 22:6n-3 seemingly compensate for the

lack of 22:6n-3 with accumulation of long chain n-6 FA

such as 22:5n-6 and biosynthetic intermediates (i.e. 22:4n-6,

20:4n-6, 20:3n-6 and 18:2n-6). This pattern was clearly

demonstrated in the first axis of the PCA analysis. The

increase of long chain and shorter chain n-6 PUFA in

phospholipids of fish fed Diet 1 led to equivalent total

PUFA levels in mitochondrial phospholipids of trout fed

the two diets. Close examination of the compositional data

from rats fed diets with low and high contents of fish oil

(Lemieux et al. 2008) shows that total PUFA, total MUFA,

total SFA and 18:1n-9 in mitochondrial phospholipids are

unchanged. Weanling rats fed either maternal diet or a diet

high in linolenic acid (18:3n-3) (Astorg and Chevalier,

1991), also drastically modified FA compositions of three

main phospholipid classes (i.e. phosphatidylcholine, phos-

phatidylethanolamine and cardiolipin) of heart and liver

mitochondria, but maintained total PUFA, total MUFA,

total SFA and 18:1n-9. These patterns suggest that fish and

mammals possess a common strategy to maintain a relative

stability of certain FA as well as total SFA, total MUFA,

total PUFA via selective incorporation and modification of

FA, presumably with the aim of regulating membrane

dynamics.

Given the importance of membrane processes in setting

basal metabolic rate, it stands to reason that the interaction

of membrane proteins with their lipid environment be reg-

ulated. Pacemaker theory argues that overall FA unsatura-

tion influences the kinetic energy of membrane proteins

thereby setting rates of membrane processes and conse-

quently basal metabolic rate (Hulbert 2007, 2008). We

found that dietary FA availability markedly influences lev-

els of major FA in phospholipids, but total SFA, total MUFA

and total PUFA remain quite stable and, in keeping with the

membrane pacemaker hypothesis, functional properties

change little. The stability of the overall characteristics of

membrane FA suggests that the physical characteristics

of the membrane, such as fluidity and dynamic phase

behaviour, would be similar in mitochondria from trout fed

the two diets. On the other hand, specific interactions

between phospholipids and membrane proteins can influ-

ence catalytic capacities, as shown by the impact of cardi-

olipin composition on the activity of complex I (Paradies

et al. 2002) and COX (Robinson 1993; Frick et al. 2010).

Thus, specific molecular species of phospholipids within the

micro-environment of enzymes may dictate functional

properties. Such specific molecular species would comprise

a small percentage of total FA in mitochondrial membranes.

It is conceivable that the preservation of specific molecular

species pass unnoticed during the marked changes of the

overall FA composition of total phospholipids.

The membrane pacemaker hypothesis puts considerable

emphasis on the influence of 22:6n-3 levels on rates of

membrane processes. The absence of such effects follow-

ing dietary modulation of 22:6n-3 levels in our experiments

(see also Guderley et al. 2008) is hard to reconcile with the

membrane pacemaker hypothesis. Fish mitochondria

maintain far higher 22:6n-3 contents than mammalian

mitochondria (Hazel 1995; Brookes et al. 1998; Hulbert

2003). Remarkably, despite being fed for several months

with a diet containing\0.1 % of 22:6n-3, trout could keep

22:6n-3 at 24 % of total FA in mitochondrial phospholip-

ids. Maintaining a species-specific ‘‘basal’’ level of 22:6n-3

in mitochondrial membranes may be critical for the

maintenance of mitochondrial function, with higher levels

having little impact. The biphasic response of 22:6n-3

levels in rat skeletal muscle phospholipids to dietary PUFA

balance and 18:3n-3 availability (Abbott et al. 2010) is

compatible with this suggestion.

While functional characteristics changed little with diet,

the growth of our trout during the lengthy period required

for our measurements revealed allometric changes of

functional properties and FA composition in trout mito-

chondria. Among the functional properties, state 4 rates,

complex I and COX activity were positively correlated with

body mass, whereas F0F1 ATPase activity decreased with

body mass. Diet influenced the allometry of Complex I,

with mass having a stronger positive impact for fish fed Diet

1. F0F1 ATPase and COX showed strong but opposite

allometric effects. Among mitochondrial phospholipids, we

found the proportions of the 24:1 series, 22:5n-3 and 22:5n-

6 to be correlated with body mass. General parameters such

as total PUFA, total MUFA, total SFA, % n-3, % n-6, n-3/n-

6 as well as major FA did not change with body mass.

Similarly, age had a greater effect on specific FA of trout

liver phospholipids than on their general parameters

(Almaida-Pagan et al. 2012). The 24:1 series was best

J Comp Physiol B

123


correlated with inter-individual variation in functional

properties, particularly of the F0F1 ATPase and COX.

These data are compatible with the hypotheses that (1) 24:1

FA are modified with body mass to regulate functional

activities and (2) the opposite allometries of F0F1 ATPase

and COX are due to modifications in the membrane envi-

ronment. The low levels of the 24:1 FA indicate that if these

hypotheses hold, the changes of 24:1 FA levels would occur

in specific phospholipid molecular species, located likely

within the micro-environment of the proteins. The 24:1 FA

also change with age in several major PL classes of trout

liver mitochondria (Almaida-Pagan et al. 2012). Interest-

ingly, F0F1 ATPase and COX show opposite responses to

modification of FA composition of mitochondrial phos-

pholipids in rat heart (Yamaoka et al. 1988). On the basis of

inspection of their data, Yamaoka and coworkers suggest

that these responses were due to replacement of 18:2n-6

with 22:6n-3 and 18:1n-9 particularly in cardiolipin. Inter-

specific comparisons show that membrane FA composition

changes with body size, as n-6 PUFA in mitochondrial

membranes increase with body size in mammals (Porter

et al. 1996; Hulbert et al. 2002a), birds (Hulbert et al.

2002b) and reptiles (Brand et al. 1991; Brookes et al. 1998).

This increase has been linked with allometric changes in

proton leak and is a key point underlying the membrane

pacemaker hypothesis. While the allometric tendencies we

report are intra- rather than inter-specific, they were

apparent over a threefold range in size and despite marked

differences in the dietary availability of FA.

Conclusion

Our central finding is that, although the FA composition of

mitochondrial membranes was strongly modified by diet,

membrane-bound processes and protein–lipid interactions

seem to have been protected as very few functional

parameters were modified. The activity of the ETC com-

plexes and the F0F1 ATPase were no more sensitive to

changes in membrane FA composition than overall mito-

chondrial properties. Second, we found that the FA that

were best correlated with inter-individual variability in

functional properties were minor and changed with body

mass, regardless of diet. This suggests that regulated and

specific changes in FA composition can be used to adjust

catalytic properties of membrane-bound enzymes, concur-

ring with the many demonstrations of regulated changes in

membrane composition and function with physiological

transitions such as thermal acclimation. Responses to

changed availability of FA in the diet were controlled in

such a fashion that the overall membrane characteristics

(total SFA, MUFA and PUFA) remained constant, with

specific FA (e.g. 18:1n-9, 22:5n-6, 22:6n-3) also showing

clear evidence of regulation. It appears that trout deprived

of a source of n-3 FA will activate production of n-6 FA,

notably 22:5n-6, presumably to compensate for the lack of

22:6n-3. The resulting stability of the functional properties

of mitochondria seems of considerable adaptive value. In

nature, the lipid composition of available foods is likely to

differ seasonally and spatially. By maintaining function

through regulated modification of FA composition of

phospholipids, trout buffer their metabolic capacities

against dietary variability. Given the high cost of the

mitochondrial proton gradient and the vital role of mito-

chondria in cellular ATP supply, regulation of FA com-

position of phospholipids may limit dietary sensitivity of

mitochondrial function, favouring the maintenance of

capacity under a range of feeding conditions.

Acknowledgments N.M. was a recipient of a FONCER grant from

Natural Sciences and Engineering Research Council of Canada and a

scholarship for the programme de stages internationaux from Fondsde recherche nature et technologies du Quebec. Many thanks are due

to the staff of the LARSA and to a special colleague for their assis-

tance with holding the fish. The authors extend their sincere thanks to

Dr. Grant W. Vandenberg and Emilie Proulx for their help with the

mixing and pelleting of experimental diets. Special thanks to Fabi-

enne Legrand for her help with phospholipid analysis. Comments

from anonymous reviewers helped to improve this paper. This work

was supported by Natural Sciences and Engineering Research Council

of Canada grant to H.G.

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