An allometric comparison of microsomal membrane lipid composition and sodium pump molecular activity in the brain of mammals and birds
1 Metabolic Research Centre, Department of Biomedical Science, University of
Wollongong, Wollongong, NSW 2522, Australia
2 Department of Biological Science, University of Wollongong, Wollongong,
NSW 2522, Australia
* Author for correspondence at present address: Garvan Institute of Medical Research, 384 Victoria Street, Darlinghurst, NSW 2010, Australia (e-mail: n.turner{at}garvan.org.au)
Accepted 10 November 2004
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Summary |
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Key words: body size, cholesterol, docosahexaenoic acid, fatty acids, Na+, K+-ATPase, phospholipids
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Introduction |
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At the cellular level, a substantial proportion of metabolic rate is
associated with the maintenance of ion gradients across membranes
(Rolfe and Brown, 1997), with
one of the most important gradients being the Na+ gradient. The
sodium pump (Na+,K+-ATPase), a ubiquitous enzyme found
in the cell membrane of all animals, is responsible for the maintenance of
this Na+ gradient. In humans and rats, the in vivo
activity of the sodium pump is estimated to account for approximately 20% of
resting metabolic rate (Rolfe and Brown,
1997
), however in tissues such as the kidney and brain, where
sodium pump concentration is the highest, it can account for as much as 60% of
resting metabolism (Clausen et al.,
1991
).
The in vitro activity of the sodium pump varies considerably
between species. For example, in addition to measuring the respiration rate of
mammalian liver and kidney slices, Couture and Hulbert
(1995b) also determined sodium
pump activity (measured as [86Rb]+ uptake), and found
that activity was higher in small mammals and declined with allometric
exponents of -0.13 and -0.14 in the liver and kidney respectively. This study
however, did not determine whether tissues in small mammals had an increased
sodium pump activity due to a greater concentration of sodium pumps or an
increased molecular activity of individual sodium pumps (i.e. turnover rate of
substrate per enzyme). Comparisons of endotherms and ectotherms have shown
that the higher metabolic rate of the laboratory rat (Rattus
norvegicus) compared to the cane toad (Bufo marinus) is
associated with a 3-4-fold greater sodium pump molecular activity in rat
tissues (Else et al., 1996
;
Else and Wu, 1999
), and it has
been proposed that changes in the molecular activity of membrane proteins are
one of the major mechanisms underlying differences in metabolism
(Hulbert and Else, 1999
).
Thus, the first aim of the current study was to determine sodium pump
molecular activity in the brain of mammals and birds of different body size,
to examine if the high mass-specific metabolic rate of small endotherms is
associated with an increased molecular activity in their sodium pumps.
In the second part of the present investigation we have examined microsomal
membrane lipid composition in the brain of the mammalian and avian species.
Changes in the fatty acid composition of neural membranes have been shown to
alter the kinetic properties of the sodium pump (Gerbi et al.,
1993,
1994
), and recently functional
reconstitution experiments involving lipid crossovers between species, have
provided direct evidence that membrane fatty acid composition is a major
determinant of the molecular activity of the sodium pump enzyme
(Else and Wu, 1999
;
Wu et al., 2004
). Accordingly,
we have analysed membrane lipid composition to determine whether variations in
sodium pump molecular activity in the brain of the mammals and birds are
underpinned by variations in membrane fatty acid composition.
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Materials and methods |
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Emus (Dromaius novaehollandiae Latham) were purchased from Marayong Park Emu Farm (Falls Creek, NSW, Australia). Zebra finches (Taeniopygia guttata Vieillot), ducks (Anas platyrhynchos L.) and geese (Anser anser L.) were purchased from local pet shops or at the Narellan Aviary Bird Auction (NSW, Australia). Feral pigeons (rock dove, Columba livia Gmelin) were from a local pigeon breeder (T. Cooper, Corrimal, NSW, Australia). House sparrows (Passer domesticus L.), starlings (Sturnus vulgaris L.), and pied currawongs (Strepera graculina Shaw) were free-living animals caught locally in the Wollongong area. Birds were either used immediately on the day of collection or were housed short-term (2-3 days) in the University of Wollongong animal house under the same environmental conditions as described for mammals, with ad libitum access to food and water. For the finches and sparrows the food was mixed birdseed, and for the ducks and geese it was a commercial mixture of pellets and seeds. The diet of the other birds before purchase was unknown. All birds were killed by lethal overdose of either Lethabarb® or Nembutal® (pentobarbitone sodium, 100 mg kg-1 body mass; intraperitoneal, except in the case of the emus where the injection was intrajugular).
Body mass and brain mass of the mice, rats and all the bird species were
obtained immediately following death. For sheep and cattle, carcass weights
were used to calculate the weight of the whole mammal, assuming carcass weight
was 55% of total body mass as is routinely used commercially
(Couture and Hulbert, 1995b).
The pig carcass weight included the skin, and since this organ accounts for
15-20% of total body mass, carcass weight was assumed to be 70% of total body
mass. Brain mass for the sheep, pigs and cattle were obtained immediately
prior to the commencement of experimental assays. All procedures were
performed in accordance with the National Health and Medical Research Council
Guidelines for Animal Research and were approved by the Animal Experimentation
Ethics committee of the University of Wollongong.
Materials
[3H]ouabain (30.0 Ci mmol-1, 37 MBq, 96.2% purity) in
1:9 toluene:ethanol was obtained from Amersham Pharmacia Biotech (Castle Hill,
NSW, Australia). Ouabain was purchased from ICN Biomedicals Inc. (OH, USA),
scintillation cocktail (Ready SafeTM) from Beckman (Gladesville, NSW,
Australia), tissue solubiliser (Soluene®-350) from Packard
Biosciences (Mt Waverley, VIC, Australia) and Na2ATP (special
quality) from Boehringer Mannheim (Mannheim, Germany). Sodium deoxycholate
(DOC), ethylenediaminetetracetic acid (EDTA), ammonium molybdate, ferrous
sulphate, analytical grade methanol, chloroform, ethyl acetate, n-hexane, and
extra pure grade diethyl ether and petroleum spirit (40-60°C) were
purchased from Merck Pty Ltd (Kilsyth, VIC, Australia).
Lethabarb® and Nembutal® (pentobarbitone sodium)
were from Boehringer Ingelheim Pty Ltd (Artarmon, NSW, Australia). Analytical
grade butylated hydroxytoluene (BHT) 14% (w/v), boron trifluoride in methanol,
stannous chloride, lab reagent sodium hydrosulfite (80%), the cholesterol
assay kit, the cholesterol calibrator and all fatty acid standards were from
Sigma Aldrich (Castle Hill, NSW, Australia). Silane-treated glass wool was
purchased from Alltech associates (Baulkham Hills, NSW, Australia).
Strata® SPE SI-2 Silica and FL-PR Florisil columns were from
Phenomenex (Pennant Hills, NSW, Australia). All other chemicals and reagents
used were of analytical grade and obtained from Ajax chemicals (Auburn, NSW,
Australia).
[3H]ouabain binding
The concentration of sodium pump sites was determined using the
[3H]ouabain binding method described by Else et al.
(1996). Brains were removed
from the animals immediately following death, cortical sections were cut into
small pieces (2-10 mg) and placed in ice-cold K+-free medium that
closely resembled the ionic composition of mammalian and avian plasma (see
below). Mammalian K+-free medium was based on a solution previously
used (Else at al., 1996
) and
included (in mmol l-1): 125 NaCl, 1.2 MgSO4, 1.2
NaH2PO4, 25 NaHCO3, 1.3 CaCl2 and
5 glucose, pH 7.4. As [3H]ouabain binding had not previously been
conducted in avian brains, two different solutions were used to assess
[3H]ouabain binding in the birds (in mmol l-1): a
K+-free medium (124 NaCl, 1.2 MgSO4, 1.1
NaH2PO4, 25 NaHCO3, 2.5 CaCl2 and
11.1 glucose, pH 7.4), and a 4.5 mmol K+-medium (125 NaCl, 3.4 KCl,
1.2 MgSO4, 1.1 KH2PO4, 25 NaHCO3,
2.5 CaCl2 and 11.1 glucose, pH 7.4). A K+ concentration
of 4.5 mmol was chosen for the incubations as it approximates the documented
average plasma K+ concentration for a large number of birds
(Altman and Dittmer, 1974
;
Prosser, 1973
).
K+-free media are generally used in [3H]ouabain binding
studies as K+ is thought to inhibit binding of ouabain to the
sodium pump (Wallick et al.,
1980
); however, Else
(1994
) demonstrated increased
levels of binding with varying levels of K+. Under the current
experimental conditions, there was no statistical difference in the measured
[3H]ouabain binding sites using the different media, and therefore
their average was used to estimate sodium pump density in birds.
Brain tissue samples were preincubated in ice-cold K+-free medium for 2x10 min periods to reduce tissue K+. The samples were then incubated in 2 ml of mammalian or avian K+-free medium containing 1 µCi ml-1 [3H]ouabain and a final ouabain concentration of 5x10-5 mol l-1. Parallel incubations containing the same amount of [3H]ouabain and a final concentration of 10-2 mol l-1 ouabain were also conducted to determine non-specific binding. Mammalian tissues were assayed at 37°C, while avian incubations were completed at 40°C. Incubations were gassed continuously for 2 h with carbogen (5% CO2, 95% O2), to maintain physiological pH levels (7.4), and to circulate the incubation medium around the tissue samples.
After incubation, the tissue samples were washed five times (8 min per
wash) in 3 ml of ice-cold K+-free medium to reduce [3H]
activity associated with non-specific sites, as previously characterised
(Else, 1994;
Else et al., 1996
). Following
the wash procedure, samples were blotted lightly, weighed (±0.01 mg)
and placed in 200 µl of tissue solubiliser (Soluene®-350)
overnight. Readysafe scintillation cocktail (2 ml) was added to each vial and
[3H] activity counted on a Wallac 1409 Liquid Scintillation Counter
(Turku, Finland) with d.p.m. correction.
[3H]ouabain binding was expressed as relative uptake, i.e. [3H] activity taken up per gram wet weight of tissue relative to [3H] activity in the incubation medium. Specific uptake was calculated following subtraction of [3H] activity determined in excess ouabain (10-2 mol l-1), which was deemed non-specific uptake. [3H]ouabain binding sites per gram of tissue were determined by multiplying the specific uptake by the total ouabain concentration in the medium. Sodium pump density was calculated assuming a 1:1 stoichiometry between sodium pump units and ouabain binding sites and was expressed as picomoles of sodium pumps per gram of brain wet mass.
Determination of Na+,K+-ATPase activity
Na+,K+-ATPase activity was determined in brain
homogenates using a modified method of that described by Esmann and Skou
(1988). Dilute homogenates
were prepared (2%, w/v) in ice-cold 250 mmol l-1 sucrose, 5 mmol
l-1 EDTA, 20 mmol l-1 imidazole (pH 7.4) using a
glass-glass homogeniser. A mild detergent treatment was applied to the samples
prior to the assay to elicit maximal Na+,K+-ATPase
activity. A 150 µl volume of homogenate was mixed under constant stirring
with 150 µl of sodium deoxycholate (1 mg ml-1) and was allowed
to stand at room temperature for 15 min. Samples (50 µl) of the detergent
treated homogenates were then preincubated in
Na+,K+-ATPase assay medium (in mmol l-1: 30
histidine, 4 MgCl2, 124 NaCl, and either 1 ouabain or 20 KCl, pH
7.5) for 10 min at 37°C (mammals) or 40°C (birds) to allow for thermal
equilibration and binding of ouabain to the sodium pumps. Enzyme activity was
initiated by the addition of 3 mmol l-1 ATP and allowed to proceed
for 5 min. The reaction was terminated by the addition of an equal volume of
perchloric acid (0.8 mol l-1) at 4°C. Inorganic phosphate (Pi)
was determined as previously described
(Else, 1994
). Maximal
Na+,K+-ATPase activity was calculated as the difference
in inorganic phosphate liberated (from ATP) in the presence and absence of 1
mmol l-1 ouabain (minus and plus KCl, respectively). Experiments
were conducted in duplicate or better.
Mammals and birds were assayed at different temperatures to approximate in vivo conditions. To allow comparison between the two vertebrate classes, thermal quotient (Q10) values for Na+,K+-ATPase activity from the brain of several birds was determined. These Q10 values were all close to 2.0 (range 1.6-2.4), and as such a this value was used to correct bird Na+,K+-ATPase activity to 37°C and therefore allow comparison with mammals.
Molecular activity
Molecular activity is defined as the maximal rate of substrate turnover by
a protein, and for the sodium pump was derived by dividing maximal
Na+,K+-ATPase activity (expressed as pmol Pi
min-1 mg wet mass-1) by the number of sodium pumps (in
pmol mg wet mass-1) for the same preparation. The net result was
expressed as the number of ATP molecules hydrolysed by each sodium pump per
minute.
Preparation of microsomal membranes
All lipid measurements were conducted using microsomal membranes prepared
from brain (cortex) homogenates (10% in 250 mmol l-1 sucrose, 20
mmol l-1 imidazole, 1 mmol l-1 EDTA; pH 7.4) that were
centrifuged at 3000 g for 3 min and a further 10 min at 10,000
g to remove nuclei and mitochondria respectively. The
supernatant was then centrifuged at 98,000 g for 35 min and
the resultant pellet, designated microsomal membranes, was resuspended in 25
mmol l-1 imidazole, 2 mmol l-1 EDTA (pH 7.5). In the
current investigation we examined microsomal membranes in preference to whole
tissue, as phospholipids isolated from microsomes (i.e. plasma membrane, golgi
and endoplasmic reticulum phospholipids) are more representative of the lipids
that would be directly surrounding the sodium pump, rather than whole tissue
phospholipids which would also contain nuclear and mitochondrial membrane
fractions. Furthermore we prepared microsomes for all species from the same
area of the brain where sodium pump determinations were conducted (i.e.
cerebral cortex), to minimise any region-related differences and to allow
direct comparison between the sodium pump and membrane lipid measurements.
Microsomal fractions were prepared from the tissue of individual animals,
except for the mouse and zebra finch where some microsomal fractions were
pooled samples from the tissue of 2-3 animals. The protein content of
microsomal preparations (and tissue homogenates) was determined by the Lowry
method, using bovine serum albumin as the standard.
Analysis of phospholipid fatty acid composition
Total lipid was extracted from the microsomal preparation by standard
methods (Folch et al., 1957)
using ultra-pure grade chloroform:methanol (2:1, v/v) containing butylated
hydroxytoluene (0.01%, w/v) as an antioxidant. Phospholipids were separated
from neutral lipids by solid phase extraction on silicic acid columns. Fatty
acid analysis of the phospholipid fraction was determined as described in
detail elsewhere (Pan and Storlien,
1993
). The cholesterol content of microsomal preparations was
determined by enzymatic assay (Sigma chemicals). Analysis of phospholipid
content was via a phosphorus assay (Mills
et al., 1984
).
Statistical analyses
All statistical comparisons were determined and tested for significance
using the mean value for each species (i.e. N=5 for mammals and
N=8 for birds). Allometric equations were determined by linear
regression (least-square method) of log-transformed values using
JMP® 4.0.1 software (SAS Institute Inc., NC, USA). All figures
were produced using KaleidaGraphTM 3.51 software (Synergy Software,
Reading, PA, USA). Allometric relationships were tested for significance using
the Pearson product moment correlation coefficient, with n-2 degrees
of freedom. Significance for all relationships was accepted at the level of
P<0.05 and all results are reported as means ± standard
error of the mean (S.E.M.).
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Results |
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Smaller species had relatively larger brains, particularly in the birds where the brain represented 3.3% of body mass in the zebra finch, but only 0.05% in the emu (Table 1). The allometric exponents describing the body-size-related variation in brain mass were 0.72 and 0.47 for mammals and birds respectively. Protein concentration (per gram of wet mass) was significantly higher in the smaller species, declining with allometric exponents of -0.02 and -0.03 in the mammals and birds respectively. As such the brains of the mice contained 17% more protein (per gram of wet mass) than those of cattle, while sparrow and starling brains contained 23% more protein than emu brains (Table 1).
Na+,K+-ATPase activity, sodium pump density, and molecular activity measured for the mammals and birds are presented in Table 2. Na+,K+-ATPase activity values were measured at 37°C for mammals and corrected to 37°C for birds (see Materials and methods). When examined relative to body mass, there was a significant decrease in Na+,K+-ATPase activity for both the mammals (P=0.05) and birds (P<0.01) (Fig. 1). Calculated from the allometric exponents (-0.06 and -0.07), for every doubling in body mass there would be a 4.1% and 4.7% decrease in Na+,K+-ATPase activity in the mammals and birds respectively.
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Sodium pump density per gram of brain wet mass was generally similar between the mammal and bird species. When considered relative to body mass (Fig. 2), there was a close to significant (P=0.056) allometric increase in sodium pump density in the larger mammals, with an exponent of 0.06 observed. In the brain of birds the trend was for a greater sodium pump density in the smaller species, although the exponent describing this relationship (-0.07) was not statistically different from zero.
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Molecular activity was calculated by dividing maximal Na+,K+-ATPase activity (at 37°C) by the sodium pump density to give the number of ATP molecules hydrolysed by each sodium pump per minute (ATP min-1). In mammals, molecular activity varied approximately threefold over the body mass range from 9000-29,000 ATP min-1, while for birds less variation was seen, with most species having a molecular activity of approximately 15,000 ATP min-1 (Table 2). When examined relative to body mass (Fig. 3) there was a significant (P=0.02) decrease in molecular activity in the mammals with body mass explaining 86% of the variability. In contrast with this, less than 1% of the variability in bird brain molecular activity values could be explained by body mass (Fig. 3).
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Table 3 presents the cholesterol and phospholipid content, along with their molar ratio, in brain microsomal membranes from the mammals and birds. No significant body-size-related variation was observed in any of the parameters presented in Table 3. Comparison of the mammals and birds shows that despite a similar microsomal cholesterol content (per mg of protein), birds have a greater molar ratio of cholesterol:phospholipid in their microsomal membranes, due to a lower content of phospholipid. From the cholesterol:phospholipid ratios it can be seen that brain microsomes from mammals would contain approximately 2-3 phospholipids per molecule of cholesterol, while in brain microsomes from birds there would be 1.0-1.5 phospholipids per molecule of cholesterol.
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The fatty acid profile of brain microsomal phospholipids for mammals and birds are presented in Table 4 and Table 5 respectively. In phospholipids from mammalian brain microsomes, there were no statistically significant allometric trends observed for fatty acid composition. The brain of all mammals displayed a relatively high content of 22:6 (n-3), with the highest levels observed in the cattle and a considerably lower amount in the pigs (Table 4). On average 68% of the fatty acid chains were unsaturated, with an unsaturation index of 265. The mean values for the other major parameters were 20.8% monounsaturated fatty acids (MUFA), 47.4% polyunsaturated fatty acids (PUFA), 18.3% n-6 PUFA, 29.1% n-3 PUFA and 27.9% 22:6 (n-3).
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In microsomal phospholipids from the avian brains there were a small number
of significant allometric trends. The content of 18:2 (n-6) and 20:3
(n-6) were significantly lower (P<0.05) in the larger
birds, which resulted in a significant allometric increase
(P<0.01) in the ratio of 20:4 (n-6): 18:2 (n-6),
which is an estimate of 5 and
6 desaturase enzyme activity. All
bird species had a high content of 22:6 (n-3) with the highest levels
found in the emu and the lowest levels in the pigeon
(Table 5). The emu and duck had
the highest levels of unsaturation (as indicated by unsaturation index), which
appeared to result from a greater content of 22:5 (n-6) in these
species, plus the high levels of 22:6 (n-3). The average values for
the major parameters were 71% total unsaturates, 314 unsaturation index, 13.2%
MUFA, 58.0% PUFA, 23.1% n-6 PUFA, 34.9% n-3 PUFA, and 34.1%
22:6 (n-3).
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Discussion |
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One of the goals of the current study was to determine if the higher Na+,K+-ATPase activity observed in small mammals and birds was the result of an increased concentration of sodium pumps, an increased molecular activity, or a combination of both. Compared with large mammals, small mammals had both a lower concentration of sodium pumps (Fig. 2) and an increased molecular activity in individual sodium pumps (Fig. 3), indicating that a combination of allometric changes in both these variables contributed to the body-size-related variation observed in Na+,K+-ATPase activity (Fig. 1). In birds there was a high degree of variation among the species and, as such, no significant relationships were found between body size and sodium pump density (Fig. 2) or molecular activity (Fig. 3). It is worth noting, however, that despite this variation, the allometric exponent measured for sodium pump density (-0.07) was equal to the exponent observed for Na+,K+-ATPase activity (Fig. 1), suggesting that changes in the concentration of sodium pumps may partly contribute to the allometric variation observed in brain Na+,K+-ATPase activity in birds.
The primary function of the sodium pump in the brain is the maintenance of
the Na+ gradient, which provides the immediate energy source for
action potentials and supports the co-transport of various compounds such as
amino acids (Clausen et al.,
1991). Additionally the sodium pump also provides the gradient for
Na+/Ca2+ exchange in the brain
(Clausen et al., 1991
). In
mammals the brain expresses a number of different isoforms
(
1,
2 and
3) that are
thought to mediate the various processes performed by the sodium pump
(Juhaszova and Blaustein,
1997
). In intact mammalian brain, in vivo sodium pump
activity has been estimated by isotope flux studies to be between 0.10-0.74
µmol ATP h-1 mg wet mass-1
(Clausen et al., 1991
). These
values represent up to 50% of brain O2 consumption, which is
evidence of the importance of the sodium pump to brain function. Compared with
the maximal in vitro activity values measured in the present study
(Table 2), it can be estimated
that in vivo the sodium pump is operating at less than 15% of
maximum, although this may vary depending on which section of the brain is
active at any particular time.
The allometric exponents for brain mass in the mammals and birds were 0.72
and 0.47, respectively. These allometric slopes are similar to previous
investigations (Else and Hulbert,
1985; Peters,
1983
) and demonstrate that as body size increases, relative brain
size decreases in both mammals and birds. Na+,K+-ATPase
activity was combined with brain mass and expressed as the micromoles of
inorganic phosphate liberated (from ATP) per brain per hour, and assuming that
this value represented the maximum rate, and using a P/O ratio of 2.0
(Rolfe and Brown, 1997
), it
was possible to determine the potential maximal daily energy expenditure by
the sodium pump (kcal day-1). When these
Na+,K+-ATPase values were compared with the basal
metabolic rates from Table 1,
it was found that potential maximal energy expenditure by the sodium pump
represented approximately 25% of BMR in small species, but only approximately
8% of BMR in large mammals and birds. It should be noted however, that it is
unknown whether all of the species are operating at a similar percentage of
these maximal in vitro Na+,K+-ATPase
activities, under in vivo basal conditions.
To examine if differences in sodium pump molecular activity were associated
with alterations in membrane lipid composition, we also determined the fatty
acid composition of microsomal membranes from the mammalian and avian brains.
Phospholipid fatty acid composition has been shown to vary with body mass in a
number of tissues (heart, skeletal muscle, kidney and liver) in mammals
(Couture and Hulbert, 1995a;
Hulbert et al., 2002b
) and
also in bird skeletal muscle (Hulbert et
al., 2002a
). Brain phospholipids from mammals however, show no
body-size-related variation (Couture and
Hulbert, 1995a
; Hulbert et
al., 2002b
), and in the current study microsomal phospholipids
from mammalian brains followed a similar pattern
(Table 4), indicating that the
lack of allometric variation is also manifest in subcellular membranes. The
relationship between body size and membrane acyl composition has never
previously been examined in avian brains, though the relative consistency of
the fatty acid profile in the bird microsomes in the present study
(Table 5), indicates that
similar trends to those seen in mammals may also exist in birds.
The consistency of the fatty acid profile in the brain of the mammals and
birds suggests that variations in membrane lipid composition are not
associated with the significant allometric decline observed in molecular
activity in the mammals or the inter-species differences observed for
molecular activity in the birds in the current study. Indeed, in contrast with
our previous work (Else and Wu,
1999; Wu et al.,
2004
), no significant correlations were observed between molecular
activity values and fatty acid composition for the current data set. The
reason for this lack of correlation is not clear, but is potentially related
to other factors such as isoform differences between species.
Of interest was the consistency of the fatty acid profile in the brain of
the mammals and birds. The exact mechanisms that maintain this constant
membrane profile in the brain of different species are unknown at present. The
composition of membranes is highly regulated and although the relative
occurrence of various fatty acids may be influenced by their presence or
absence in the diet, it is difficult to substantially alter phospholipid fatty
acid composition through dietary manipulation. The main parameter that appears
to be affected by diet is the relative percentage of n-6 and
n-3 PUFA in the membrane (Hulbert
et al., 2005), and interestingly the phospholipids from the sheep,
cattle and geese all had higher levels of 22:5 (n-3) compared with
the other species (Tables 4 and
5), which may indicate that
these animals were pasture-fed, as forage crops are known to contain high
levels of n-3 PUFA (Christie,
1981
), and other tissues from these animals also contained high
levels of long-chain n-3 PUFA (N.T., A.J.H. and P.L.E.,
unpublished).
Despite the relative consistency in fatty acid profile, there were a small
number of significant allometric trends observed in the brains of birds,
including a decrease in the content of 18:2 (n-6) and 20:3
(n-6), along with a significant increase in the ratio of 20:4
(n-6):18:2 (n-6) in the larger species. In mammals, the
ratio of 20:4 (n-6):18:2 (n-6) gives an estimate of the
activity of the 5 and
6 desaturase enzymes and the increased
activity observed in the larger birds, coupled with the reduced levels of both
18:2 (n-6) and 20:3 (n-6) indicates a very active conversion
of short-chain n-6 PUFA to their long-chain derivatives. This was
very evident in the duck and emu where the highest ratio of 20:4
(n-6):18:2 (n-6) and also the highest levels of 22:5
(n-6) were seen (Table
5). Interestingly, while the estimated desaturase activity was
increased in the larger birds, the elongase enzyme activity (estimated from
the 18:0: 16:0 ratio) was reduced in these species (results not shown).
Whether elongase/desaturase enzyme systems operate in birds as they do in
mammals is unknown, as within the mammals there was no allometric trend seen
in the ratio of 20:4 (n-6): 18:2 (n-6), while the ratio of
18:0:16:0 was actually increased in the larger mammals.
Comparison of the mammals and birds showed that the major differences were
a greater content of 18:1 (n-9) in the mammals and higher levels of
both 22:5 (n-6) and 22:6 (n-3) in the birds. Farkas et al.
(2000) also found higher PUFA
levels in birds compared to mammals and of particular interest in the current
study was the high levels of 22:5 (n-6) observed in all of the birds
(Table 5). Within the mammals
the pig had the highest levels of 22:5 (n-6) along with the lowest
levels of 22:6 (n-3) (Table
4). These fatty acids appear to be regulated in a reciprocal
manner in the brain of mammals, where 22:6 (n-3) is the preferred
long chain PUFA, but can be partially compensated for by 22:5 (n-6),
if there is a dietary deficiency of n-3 PUFA
(Carrié et al., 2000
;
Sheaff et al., 1995
). While
these fatty acids are similar in structure, recent work suggests that the
additional double bond present in 22:6 (n-3), has a major impact on
its physical properties, and as a result 22:5 (n-6) may not
compensate functionally for 22:6 (n-3) with regards to lipid-protein
interactions in the membrane (Eldho et
al., 2003
). The reason for the high levels of 22:5 (n-6)
in the birds in the current study is unclear, however, it appears that the
reciprocal relationship between 22:6 (n-3) and 22:5 (n-6)
may not also be present in birds, as the highest levels of both fatty acids
were found in the emu (Table
5).
Although there were a couple of small differences in fatty acid composition
between the two vertebrate classes, it is clear that there is a specific
functional requirement for high concentrations of 22:6 (n-3) in the
brain of both mammals and birds. Indeed 22:6 (n-3) appears to be
prevalent in the brain of most vertebrates
(Else and Wu, 1999;
Farkas et al., 2000
;
Hulbert et al., 2002b
). The
high concentrations of this fatty acid in neural membranes is maintained both
by astrocytes, which actively desaturate and elongate n-3 precursors
and release the 22:6 (n-3) for uptake by the neurons
(Moore, 1993
), and by the
neurons themselves, which preserve membrane 22:6 (n-3) in preference
to other PUFA (Kim et al.,
1999
). Reductions in neural membrane 22:6 (n-3) have been
linked with a number of functional deficits, both during developmental periods
(Horrocks and Yeo, 1999
) and
during adulthood (Fenton et al.,
1999
; Hibbeln,
1998
,
2002
). While the exact
membrane property related to 22:6 (n-3) that regulates neurological
function is yet to be determined, it is potentially related to its influence
on membrane proteins such as the sodium pump
(Turner et al., 2003
).
Another interesting finding in the current study was the high molar ratio
of cholesterol:phospholipid observed in brain microsomal membranes from the
mammals and particularly the birds (Table
3). These values were much higher than those seen in the kidney
and heart of these animals (N.T., A.J.H. and P.L.E., unpublished), which is a
trend that has been shown in other investigations
(Wu et al., 2001;
Yeagle, 1985
). Cholesterol
metabolism in the brain is quite complex, and recently it was suggested that
cholesterol may be an essential factor in the formation and functioning of
synapses (Pfrieger, 2003
).
Support for this hypothesis comes from studies which show that cholesterol
turnover is much lower in the synaptic membranes of adults rats, compared with
those of young rats, where presumably a large number of synapses are being
established (Ando et al.,
2002
). Developing neurons synthesise their own cholesterol,
however it is thought that neurons in adult brains derive their cholesterol
from astrocytes (Pfrieger,
2002
), which may in part explain the high levels of cholesterol
observed in the present study, as astrocytes are the brain's most abundant
cell type.
A potential functional explanation for the high level of cholesterol, is
that it has been implicated as a major factor in the formation of lipid rafts.
Lipid rafts represent membrane microdomains where saturated phospholipids,
sphingomyelins and cholesterol aggregate and form less mobile, gel-like areas,
with PUFA-rich phospholipids maintaining a very fluid environment in the
remaining membrane (Simons and Ikonen,
1997). In the brain, lipid rafts are thought to be important in
determining the appropriate distribution and orientation of post-synaptic
receptors (Tsui-Pierchala et al.,
2002
). As mentioned above, brain phospholipids contain high levels
of PUFA, particularly 22:6 (n-3) and to a lesser extent 20:4
(n-6), however cholesterol is relatively insoluble in these fatty
acids (Brzustowicz et al.,
2002a
,b
).
Thus it is somewhat perplexing that in the brain there are high levels of both
cholesterol and PUFA, but it has been suggested that this composition may
facilitate sterol-lipid interactions in neural membranes, that promote lateral
heterogeneity and the formation of functionally important lipid rafts
(Huster et al., 1998
). It is
unknown at this stage whether lipid rafts influence the activity of the sodium
pump, however, since both cholesterol and PUFA appear to be supplied to the
neurons by astrocytes (Kim et al.,
1999
; Moore, 1993
;
Pfrieger, 2002
), the exact
importance of these glial cells in maintaining the appropriate neural membrane
lipid composition requires further investigation.
Abbreviations
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Acknowledgments |
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