R. K. Porter, A. J. Hulbert and M. D.

Brand
Am J Physiol Regulatory Integrative Comp Physiol 271:1550-1560, 1996. You might find this additional information useful... This article has been cited by 16 other HighWire hosted articles, the first 5 are: Mitochondrial adaptations to steatohepatitis induced by a methionine- and choline-deficient diet C. Romestaing, M.-A. Piquet, D. Letexier, B. Rey, A. Mourier, S. Servais, M. Belouze, V. Rouleau, M. Dautresme, I. Ollivier, R. Favier, M. Rigoulet, C. Duchamp and B. Sibille Am J Physiol Endocrinol Metab, January 1, 2008; 294 (1): E110-E119. [Abstract] [Full Text] [PDF] Adenine nucleotide translocator promotes oxidative phosphorylation and mild uncoupling in mitochondria after dexamethasone treatment M. Arvier, L. Lagoutte, G. Johnson, J.-F. Dumas, B. Sion, G. Grizard, Y. Malthiery, G. Simard and P. Ritz Am J Physiol Endocrinol Metab, November 1, 2007; 293 (5): E1320-E1324. [Abstract] [Full Text] [PDF]
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Mitochondrial metabolism during daily torpor in the dwarf Siberian hamster: role of active regulated changes and passive thermal effects J. C. L. Brown, A. R. Gerson and J. F. Staples Am J Physiol Regulatory Integrative Comp Physiol, November 1, 2007; 293 (5): R1833-R1845. [Abstract] [Full Text] [PDF] Life and Death: Metabolic Rate, Membrane Composition, and Life Span of Animals A. J. Hulbert, R. Pamplona, R. Buffenstein and W. A. Buttemer Physiol Rev, October 1, 2007; 87 (4): 1175-1213. [Abstract] [Full Text] [PDF] Cold-induced alterations of phospholipid fatty acyl composition in brown adipose tissue mitochondria are independent of uncoupling protein-1 A. Ocloo, I. G. Shabalina, J. Nedergaard and M. D. Brand Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2007; 293 (3): R1086-R1093. [Abstract] [Full Text] [PDF] Medline items on this article's topics can be found at http://highwire.stanford.edu/lists/artbytopic.dtl on the following topics: Biochemistry .. Fatty Acids Biochemistry .. Palmitate Biochemistry .. Stearate Biochemistry .. Phosphogycerides Veterinary Science .. Mammalia Medicine .. Body Mass Additional material and information about American Journal of Physiology - Regulatory, Integrative and Comparative Physiology can be found at: http://www.the-aps.org/publications/ajpregu

This information is current as of May 18, 2009 .

The American Journal of Physiology - Regulatory, Integrative and Comparative Physiology publishes original investigations that illuminate normal or abnormal regulation and integration of physiological mechanisms at all levels of biological organization, ranging from molecules to humans, including clinical investigations. It is published 12 times a year (monthly) by the American Physiological Society, 9650 Rockville Pike, Bethesda MD 20814-3991. Copyright 2005 by the American Physiological Society. ISSN: 0363-6119, ESSN: 1522-1490. Visit our website at http://www.the-aps.org/.

Allometry membrane

of mitochondrial proton leak: influence of surface area and fatty acid composition

RICHARD K. PORTER, A. J. HULBERT, AND MARTIN D. BRAND Department of Biochemistry, University of Cambridge, Cambridge CB2 lQW, United Kingdom; Department of Biochemistry, Z=-inity College Dublin, Dublin 2, Republic of Ireland; and Department of Biological Sciences, University of Wollongong, Wollongong, New South Wales 2522, Australia
Porter, Richard K., A. J. Hulbert, and Martin D. Brand. Allometry of mitochondrial proton leak: influence of membrane surface area and fatty acid composition. Am. J. Physiol. 271 (Regulatory Integrative Comp. Physiol. 40): Rl&O-R1560, 1996.--We investigated why liver mitochondria from small mammals are leakier to protons than those from larger mammals. Sixty-nine percent (+23%) of the proton leak differences appeared to relate to membrane area (less inner membrane surface area in larger animals); any residual differences must reflect differences in membrane properties. There were differences in phospholipid fatty acid composition; unsaturation index, monounsaturates, palmitate (16:0), stearate (l&O), docosahexaenoate [22:6(n-3)], and the 22:6(n-3)/22:5(n-3) ratio all correlated with body mass. Proton flux per square centimeter did not correlate significantly with body mass or, in general, with phospholipid fatty acid composition, suggesting little role for fatty acid composition in determining proton leak in mammals of different body mass. However, unsaturation index and n-3 polyunsaturated fatty acid content correlated significantly with proton leak per milligram phospholipid when literature data from reptiles and rats in different thyroid states were included, giving some support to suggestions of a general role for phospholipid fatty acid composition in determining mitochondrial proton leak. oxygen consumption; basal metabolic rate

DLJRING

OXI

DATIVE

PHOSPHORYLATION

oxygen

consump-

tion is used to create an electrochemical gradient of protons across the mitochondrial inner membrane. However, because mitochondria are not completely impermeable to protons, there is always a futile cycle of outward proton pumping and inward proton leak across the inner membrane. This leak of protons can explain the imperfect coupling of oxygen consumption to ATP synthesis (4, 17). Proton leak is a significant contributor (-25%) to the resting oxygen consumption of hepatocytes isolated from a range of mammals (4, 30) and has been estimated to be a significant contributor (-25%) to basal metabolic rate in a whole rat (4,32). Factors that determine basal metabolism affect mitochondrial proton leak. Hyperthyroidism, which increases metabolic rate, increases proton leak in hepatocytes (20) and in isolated liver mitochondria (19). Hypothyroidism, which decreases metabolic rate, decreases proton leak in liver cells (20) and in isolated liver mitochondria (18, 22). Similarly, reptiles have lower standard metabolic rates than mammals of the same mass at the same temperature, and hepatocytes and liver mitochondria from a reptile have reduced proton leak compared with a mammalian control (5).
R1550 0363-6119/96 $5.00 Copyright o 1996

In addition, there are allometric differences in proton leak. Metabolic rate per unit mass in animals is proportional to iWa2 (where A4 is body mass) and decreases lo-fold with a lO,OOO-fold increase in body mass (8,25). For example, the calculated metabolic rate of a 20-g mouse is -390 times less than that of a 340-kg horse, but its mass-specific metabolic rate is -11.4 times greater than that of the horse. Differences in massspecific metabolic rate are due partly to differences in the proportion of metabolically active organs (16). They are also due to differences in the metabolic activity of these organs (2); tissue slices from large mammals consume less oxygen than those from smaller mammals (13, 26), and liver cells from large mammals are less metabolically active than those from smaller mammals (29). The decrease in the oxygen consumption rate of cells from larger animals is due to lower numbers of mitochondria per cell and to a lower activity of these mitochondria in situ, including a lower proton leakiness (30). The proton leak in isolated liver mitochondria also decreases with increasing body mass, with an allometric exponent of - 0.14 (28). The proton leak rate per milligram of mitochondrial protein at a driving potential of 170 mV is approximately fourfold greater in liver mitochondria from a 20-g mouse than from a 340-kg horse (28). So not only is proton leak a significant contributor to the resting oxygen consumption of animals, tissues, and cells, but the decreased proton leak seen in hepatocytes and in isolated liver mitochondria from bigger animals contributes to the decrease in resting oxygen consumption observed in liver cells from mammals of greater body mass (30). Two main hypotheses have been put forward to explain differences in the proton leakiness of isolated mitochondria. Differences in proton flux per milligram of mitochondrial protein at 37C and at defined driving potential could be due to differences in the surface area of the mitochondrial inner membrane per milligram protein, resulting in the same leak rate per square centimeter of inner membrane but different leak rates per milligram protein. Alternatively, the properties of the inner membrane could be different in some way, resulting in changed leak rates per square centimeter of inner membrane as well as per milligram protein. There is a marked correlation between proton leakiness and the fatty acid content of mitochondrial phospholipids in different thyroid states and in animals of different phylogeny. This has led to the suggestion that increased unsaturation index and content of n-3 polyunsaturated fatty acids and decreased linoleate [18:2(nthe American Physiological Society

Downloaded from ajpregu.physiology.org on May 18, 2009

MITOCHONDRIAL

PROTON

LEAK

AND

BODY

MASS

RX551

G)] content in mitochondrial phospholipids may cause increased proton leak (4-7, 21). The fatty acid content of total tissue phospholipids from several tissues, including liver, changes with body mass, and this correlates with plasma membrane cation permeability (12, 13), suggesting that the same correlation of composition with body mass might be present in mitochondria from mammals of different sizes and might underlie the observed changes in mitochondrial proton leak. In this paper we investigate the mechanisms responsible for the different proton leakiness of liver mitochondria from different sized mammals. We show that the differences can be mostly explained by differences in mitochondrial inner membrane surface area/matrix volume ratio. Although there are significant changes in the nature of the fatty acids of the mitochondrial phospholipids as body mass changes, most of these changes are not significantly correlated with the differences in proton leak. However, there is a weak correlation between the unsaturation index and proton leak per milligram phospholipid, and this correlation becomes statistically significant when literature data from reptiles and rats in different thyroid states are included in the analysis.
METHODS Isoln tiorl of mitochondria. Liver tissue from all mammals was acquired fresh. Mice, hamsters, and rats were killed by cervical dislocation; other mammals were injected with a lethal dose of pentobarbitone sodium. Liver tissue was placed in ice-cold medium containing 250 mM sucrose, 10 mM tris( hydroxymethyl)aminomethane HCl, and 1 mM EGTA (brought to pH 7.4 at 200. Individual mitochondrial preparations were from eight pooled mouse livers, six pooled hamster livers, one rat liver, or portions of liver lobes from the other species. Gall bladders were removed from mouse and hamster livers before excision. Mitochondria were isolated by differential centrifugation (9). Protein was assayed by the biuret method in the presence of 50 g/l sodium deoxycholate (5). Mitochondrial suspensions were kept on ice. Measurement of mitochondrial oxygen consumption and rncm hranc potential. Mitochondria (3 mg protein) were incubated at 37C in 3 ml medium containing 120 mM KCl, 5 mM N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES)KOH, pH 7.2, and 1 mM ethylene glycol-bis(P-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA) in the presence of 7.5 mM succinate (K-+ salt), 5 uM rotenone, nigericin (100 pmol/mg protein), oligomycin (1 ug/ml), and 5 uM methyltriphenylphosphonium (TPMP). For proton leak kinetics, oxygen consumption rates and membrane potentials were titrated with O-10 mM malonate (K+ salt). Each determination was made in either triplicate or quadruplicate. Membrane potentials were measured with the use of a TPMP electrode. Proton leak rate was calculated from the oxygen consumption rate by assuming six protons were pumped (and leaked) per a tom of oxygen. State IV oxygen consumption rate was defined as the oxygen consumption under the aforementioned incubation conditions in the absence of malonate. State III uncoupled oxygen consumption rate was defined as the aforementioned incubation conditions in the absence of malonate and in the presence of carbonyl cyanidep-trifluoromethoxyphenylhydrazone (FCCP, 100 pmol/mg protein). The respiratory control ratio is the state III uncoupled rate divided by the state IV rate.
l

Preparation of mitochondria for electron microscopy. Essentially, this followed the procedure of Else and Hulbert (15) for tissue samples. Mitochondria (3 mg protein) were incubated in a minifuge tube containing 1 ml of 120 mM KCl, 10 mM HEPES-KOH, pH 7.0, 1 mM EGTA, 100 pmol/mg nigericin, 5 pM rotenone, 5 mM succinate (K+ salt), and 20 g/l glutaraldehyde for 2 h on ice. The suspension was centrifuged at 15,000 g for 2 min, then the pellet was washed three times in 0.05 M sodium cacodylate buffer, pH 7.3, postfixed in 20 g/l osmium tetroxide for 1 h, then washed three times (5 min each). It was then dehydrated in an ethanol series (50% - 100% dry) over 3 h. The sample was then infiltrated while swirling on a rheostat with Spurr epoxy resin-loo% alcohol (l:l, wt/vol) for 1 h, Spurr epoxy resin-loo% alcohol (2:1, wt/vol) for 12 h, and then 100% resin with two changes for 24 h. Samples were embedded in polymerization capsules (TAAB Laboratories Equipment, Berkshire, UK), placed in pure resin for 1 h, and cured in fresh resin at 60C overnight. Thin sections of -70-90 nm were cut with the use of a Sorvall Porter-Blum MT2 ultramicrotome using glass knives and were floated onto and dried on 200-urn copper mesh grids. The sections were stained with 20 g/l uranyl acetate for 30 min and poststained for up to 2 min with Reynolds lead citrate solution. Glutaraldehyde-fixed pellet volume was measured in parallel by including 3H20 in the original suspension as in Ref. 6. Measurement of mitochondrial inner membrane surface area. This was essentially as described by Weibel(37), using a Philips EM300 electron microscope at 80 kV accelerating voltage. Twenty sites on two copper mesh grids containing single-layered sections were randomly selected for production of hardcopy electron micrographs at a magnification of X 32,500 for each mitochondrial pellet (1 for each preparation of mitochondria). So, for instance, the value of inner membrane surface area for rat in Table 1 is based on seven pellets from seven preparations of rat liver mitochondria, i.e., a total of 140 electron micrographs. Cristae membrane surface area densities (surface/pellet volume ratios, Sv> were determined by counting the number of intersections per centimeter (I) of the membranes with 24 diameters of known length, which were marked on a transparency placed on the electron micrograph. S, was calculated using the relationship S, = 21. Measurement of the glutaraldehyde-fixed mitochondrial pellet volume (H20 pellet space in ul/mg protein) allowed inner membrane surface area to be expressed as square centimeters per milligram mitochondrial protein, and division by mitochondrial volume per milligram protein from radiolabel distribution in unfixed mitochondria (below) allowed inner membrane surface area to be expressed as square centimeters per microliter matrix volume under the conditions used in the experiments to measure proton leak rate. Phospholipid fatty acid analysis. The fatty acid composition of mitochondrial phospholipids was determined as in Ref. 23. Essentially, total lipid was extracted from mitochondrial preparations with chloroform/methanol (2: 1, vol/vol) containing butylated hydroxytoluene (0.1 g/l) (antioxidant), and phospholipids were separated by silicic acid column chromatography. Phospholipid fatty acids were methylated using BF3 in methanol. Methyl esters were separated on a BPX70 fused silica capillary column (SGE Scientific, Charles St. Ryde, NSW, Australia) in a Varian 3,300 gas chromatograph and quantified with a Shimadzu C-R3A chromatopac integrator. Fatty acids were identified by comparison with known standards and are expressed as mole percent of total fatty acids. Unidentified fatty acids represented between 1 and 7% of the total fatty acids analyzed depending on the mammal. The protective effect of butylated hydroxytoluene is emphasized by the observation that its omission from the lipid

Downloaded from ajpregu.physiology.org on May 18, 2009

R1552 Table 1. Properties

MITOCHONDRIAL

PROTON

LEAK

AND

BODY

MASS

of liver mitochondria

isolated

from mammals
Dutch Rabbit (Oryctolagus cuniculus) Mixture (3)

of different
New Zealand Rabbit (Oryctolagus cuniculus) Female 5.06

mass

Mouse (Mus tr1 usculus Gender Body mass, kg. Proton leak rate at 170 mV, nmol H smin I. mg protein I Uncoupled rate of oxygen consumption, nmol 0,srnin I. mg protein I Respiratory control ratio, (state III uncoupled/ state IV) Mitochondrial inner membrane surface area, cmz/mg protein Mitochondrial matrix volume, pI/nig protein Mitochondrial inner membrane surface area, cmn/pl matrix Phospholipid: protein ratio, m&w Females 0.02 +I0.00

Hamster (Merocricetus auratus) Females

Rat (Rattus norvegicus) Females (18) 0.25 ? 0.00 (21)

Ferret (Mustela fur01 Males 1.00 + 0.20

Sheep (Ovis aries) Females (1)

Pig Gus domesticus) Females 53.Ok3.00 (3)

Horse (Equus caballus) Females 340-i-(2)

(32)

0.10 -t 0.00

2.5 t 0.1 (3)

414(3)

129 (3)

211 (21)

218 (3)

77 (3)

72 (1)

93 (3)

175 ! 30 (3)

113+18(3)

161+5

(19)

160t36(3)

113+9(3)

77(l)

225+50(3)

306+68(3)

133%15(2)

Downloaded from ajpregu.physiology.org on May 18, 2009

2.3 ~10.4 (3)

4.520.7

(3)

4+1(19)

3.6~0.8

(3)

5.150.4

(3)

4.0 (1)

9.022.0

(3)

9?2

(3)

7k-2

(2)

500+60(3)

260~20(3)

510+40(7)

400+50(3)

160?20(2)

ND

138+6(3)

370+30(3)

170+20(2)

0.9 -e 0.1 (3)

0.9t0.2

(3)

0.97+0.08

(21)

1.220.3

(3)

0.7+0.1(3)

0.8 (1)

0.8 + 0.1 (3)

1.120.3

(3)

l.OtO.1(2)

556 -t 91(3)

289?68(3)

526?60(7)

333+93(3)

229+43(2)

ND

173+23(3)

336+96(3)

170-'26(2)

0.13 -+ 0.04

(4)

0.07~0.01

(3)

0.10t0.01(4)

0.085~0.003(3)

0.075~0.008(3)

0.08

(1)

0.07 + 0.01

(3)

0.061+

0.009

(3)

0.049

(1)

Values are expressed means 2 SE [no. of animals (*) or no. of determinations hamsters, and rats were killed by cervical dislocation; all other mammals were were lactating and all horses were pregnant. ND, not determined. TApproximate

in parentheses]. injected with a lethal value.

All mammals were dose of pentobarbitone

fed ad libitum. Mice, sodium. All sheep

extraction procedure increased the unidentified fatty acid content to 23%. Phosphorus analysis. The amount of phospholipid was determined by phosphorus analysis of the extracted total lipid essentially as in Ref. 1. Thirty microliters of 100 g/l Mg(N0&6Hz0 in 95% (vol/vol) ethanol were added to 100 pg of the total extracted mitochondrial lipid in a Pyrex glass test tube. The material was dried by shaking the tube over a strong flame until brown fumes were no longer produced. Three ashings were required for complete extraction. The tube was then allowed to cool, and 300 pl of 0.5 N HCl was added; the tube was capped with a marble and heated in a boiling water bath for 15 min to hydrolyze to phosphate any pyrophosphate formed in the ashing. After the tube had cooled, 0.6 ml of HZ0 was added. Then 1.4 ml of a mixture of one part 100 g/l ascorbic acid to six parts 42 g/l ammonium molybdate l 4HzO in 1N HzS04 was added, and the mixture

was incubated at 37C for 1 h. Absorbance was read at 820 nm against a water blank, and values, in micrograms, were determined from a standard curve. Total phospholipid (pg in sample) was calculated by multiplying micrograms of phosphorus by 780 (average molecular weight of a phospholipid) and dividing by the atomic weight of phosphorus. The greatest differences in phospholipid fatty acid composition were found between mouse and horse liver mitochondria. The mean molecular weights of the fatty acids from gas chromatography was calculated for mouse and horse liver mitochondria and were not very different. Therefore, the average molecular weight of a phospholipid was considered suitable for calculations of total phospholipid mass in liver mitochondria from all mammals used. Mitochondrial proton leak rate and membrane potential measurements. Mitochondrial proton leak kinetics were determined at 37C as described in Ref. 28. Mitochondrial volume

MITOCHONDRIAL

PROTON

LEAK

AND

BODY

MASS

R1553

and TPMP binding corrections were determined according to Ref. 3. Proton leak and membrane potential measurements were carried out within 6 h of preparation of mitochondria. On each day, proton leak and membrane potential measurements were made using liver mitochondria from rat and one (or two) other species. This ensured that any systematic error in the apparatus would not have gone unnoticed. The maximum duration of each titration, and hence the maximum time for which the mitochondria were incubated at 37C was 7 min. Mammals within a given species or breed were of a similar mass and age although the relative ages (in terms of their lifespan) may have differed between species. However, we have evidence that age does not affect proton leak in liver mitochondria within a single species: the proton leak kinetics of liver mitochondria isolated from young (180 g) and old (410 g) male rats are not significantly different (L.-F. Chien, J.A. Buckingham, and M.D. Brand, unpublished observation). Statistical analysis. Linear regressions were tested for significance using critical values of r, the Pearson product moment correlation coefficient, with n - 2 degrees of freedom, where n was the number of different species. Materials. TPMP-iodide was from Aldrich Chemical, Gillingham, Dorset, UK. Succinic acid and potassium chloride were from Fisons Chemical Equipment, Loughborough, UK. Uranyl acetate, sodium cacodylate, and sucrose were from British Drug Houses, Poole, Dorset, UK. Spurr resin, glutaraldehyde, and osmium tetroxide were from Agar Scientific, Essex, UK. Radioisotopes were from Amersham International, Amersham, UK, except for [3H]TPMP-bromide (35 Ci/mmol), which was from New England Nuclear (DuPont de Nemours), Dreieich, Germany. Solvents for fatty acid analysis were of nanograde purity and were from Mallinckrodt, St. Louis, MO. Individual fatty acid methyl ester standards were from Sigma, whereas mixtures of standards were from Supelco, Castle Hill, NSW, Australia, and Larodan Fine Chemicals, Malmo, Sweden. Other materials were obtained from Sigma Chemical, Poole, Dorset, UK. Diets. All mammals were fed ad libitum. Mice and hamsters were fed Quest 41B; rats and rabbits were fed Special

Diet Services economy rat and rabbit maintenance diet. Ferrets were on a diet of Spillers Savour (beef cat food) plus diet A(E) from Special Diet Services. Special Diet Services are supplied through Lillico, Betchworth, Surrey, UK. Sheep were fed Bearts no. 6 nuts and straw. Pigs were fed 310 Ultra-wean pellet, Dalgety Agricultural, Bury St. Edmunds, Suffolk, UK. Horses were fed Baileys no. 7 stud mix. Bearts and Baileys foods are supplied by Frenchs Mill, Cambridge, UK. None of the diets were supplemented with any particular fatty acids. RESULTS

Proton leak rate per milligram mitochondrial protein and its relationship to body mass. Mitochondria were isolated from liver of mammals over a 17,000-fold range in body mass from 20-g mice to 340-kg horses. The proton leak rate (per mg protein) across the mitochondrial inner membrane was measured as a function of the mitochondrial membrane potential, the driving force of the leak, as in Ref. 28 (not shown). The mitochondrial potential within liver cells from these species varies with body mass, from 133 mV in mouse cells to 210 mV in sheep cells (30). However, to make meaningful comparisons of proton leak we need to compare leak rates at the same driving potential. The proton leak rates at a mitochondrial membrane potential of 170 mV were interpolated from the leak kinetic graphs (Table 1) and plotted against body mass on a log-log plot (Fig. 1A). There was a significant negative correlation between body mass and the mitochondrial proton leak rate per milligram protein at 170 mV with exponent -0.129 t 0.049 (SE). These observations form part of an overlapping set with our earlier results and support them (28). Maximal mitochondrial respiratory chain activity and its relationship to body mass. The maximal chain activity of the liver mitochondria from the different

Downloaded from ajpregu.physiology.org on May 18, 2009

B
1000 I 1

y = 150.7~-~**~~,

r = -0.705,

p = 0.034

l 0 100 i----0 00 0 a

101
0.01

0.1

10

100

1000

y = 1823x-0.054,

r = -0.412, n.s. I

y = 0.531~-~~~~~, r = -0.319, n.s.

Fig. 1. Mitochondrial proton leak at 170 mV expressed in different ways as a function of body mass. A: proton leak rate per mg mitochondrial protein; B: proton leak rate per ul mitochondrial matrix volume; C: proton leak rate per mg mitochondrial phospholipid; D: proton leak rate per cm2 mitochondrial inner membrane surface area. Data calculated from Table 1. Lines were fitted using linear regression.

0.1

body mass (kg)

body mass (kg)

R1554

MITOCHONDRIAL

PROTON

LEAK

AND

BODY

MASS

y = 4.416~~~1~1,

r = 0.850, p = 0.004

r-4

0.01

0.1 hod; Inas~(kg)

100

1000

Fig. 2. Respiratory control function of body mass. Data regression.

ratio from

(state III uncoupled/state Table 1. Line was fitted

using

IV) as a linear

sized mammals was assessed by measuring oxygen consumption in the presence of excess uncoupler, FCCP (Table 1). N o significant correlation was observed between maximal chain activity per milligram protein and body mass (exponent 0.02, r = 0.184, n = 9, NS). A plot of respiratory control ratio (state ZZZ uncoupled rate/state IV rate) against body mass (Fig. 2) gave a significant correlation: larger mammals yielded more coupled liver mitochondria as expected, because larger animals yielded mitochondria with similar uncoupled rates but lower proton permeability. Mitochondrial inner membrane surface area and its relationship to body mass. Measurements of mitochondrial inner membrane surface area/matrix volume ratio were made using electron microscopy and radiolabel distribution (Table 1). Figure 3A shows that there was a significant negative correlation between surface area (cm2/ul matrix) and body mass, with exponent - 0.102 ? 0.035 (SE). Values were between 555 in mouse and 170 cm2/u1 matrix in horse liver mitochondria. The glutaraldehyde-fixed mitochondrial pellets that were used for electron microscopy contained a known

amount of protein. Measurement of the volume of these pellets using 3Hz0 allowed calculation of the surface area of the inner membrane per milligram mitochondrial protein (Table 1). Figure 3B shows that there was a weak correlation (P = 0.1) between inner membrane surface area per milligram protein and body mass, with exponent -0.096 5 0.051 (SE). The value for rat liver mitochondria (510 cm2/mg protein) is comparable to other values in the literature, e.g., 521(34) and 651(5). State IV mitochondrial matrix volume (ul/mg mitochondrial protein) measured independently with 3H20 and [ 14C] sucrose (Table 1) did not correlate significantly with body mass (exponent 0.01, r = 0.072, n = 9, NS). Mitochondrial phospholipid content and its relationship to body mass. A second measure of membrane area can be obtained from the total amount of phospholipid extractable from isolated mitochondria. This measure (Table 1) gives a value proportional to the combined bilayer area of the inner and outer membranes, assuming complete phospholipid extraction. A log-log plot of phospholipid/protein ratio against body mass (Fig. 30) gave a significant correlation with exponent -0.075 t 0.018 (SE). Division by matrix volume per milligram protein allowed calculation of milligram phospholipid per microliter matrix volume, which also correlated significantly with body mass with exponent -0.079 t 0.027 (SE) (Fig. 3C). Thus two different measures of surface area in isolated mitochondria give a significant negative correlation with body mass in mammals. Mtochondrial phospholipid fatty acid content and its relationship to body mass. Total mitochondrial phospholipid fatty acid analysis was carried out on liver mitochondria from the different mammals. The subgroupings of these fatty acids are listed in Table 2. The amounts of each specific fatty acid from the phospholipids are given in Table 3. All values are expressed as

Downloaded from ajpregu.physiology.org on May 18, 2009

A
y = 324.9~-*~~, r = -0.761, p = 0.028

Q) 10000 -5 2s &EL

y =

302.2x-oso96, r = -0.614,

p = 0.106

Fig. 3. Measures of mitochondrial surface area as a function ofbody mass. A: mitochondrial inner membrane surface area per 111matrix volume; B: mitochondrial inner membrane surface area per mg mitochondrial protein; C: mg mitochondrial phospholipid per ul matrix volume; I): mg mitochondrial phospholipid per mg mitochondrial protein. Data calculated from Table 1. Lines were fitted using linear regression.

aJ y = o.og()x-o-o79,

r = -0.739, p = 0.023

0 L

1 y = 0.083x-0*075,
r = -0.851, p = 0.004

0.01

0.1

10

100

1000

body mass (kg)

body mass (kg)

MITOCHONDRIAL

PROTON

LEAK

AND

BODY

MASS

R1555 isolated

Table 2. General properties from different mammals

Mouse 0.02 66.0 + 0.00 t 2.0 (3)

of fatty acids from phospholipids

of liver mitochondria
Dutch Rabbit New (2) Zealand Rabbit 5.06 65.5 10.1 49.3 6.0 169.1 0.34 18.30 (1)

Hamster 0.10 + 0.00 (3)

Rat 0.25 .!- 0.00 60.8 + 0.8 7.1-+0.4 54.0 + 1.0 16.8 + 0.8 223.0 2 4.0 1.54 -+ 0.06 18.86 t 0.03 (9)

Ferret 1.00 IL 0.20 69.0 -+ 1.0 12.0 + 1.0 57.0 k 2.0 13.0 + 3.0 214.0 2 10.0 0.80 2 0.20 18.70 * 0.10 (3) 2.35

Sheep 33.0 + 0.0 (3) 66.0 + 3.0 40.00 63.0

Pig + 5.00 (2) + 3.2

Horse 340 (2) 71.3 5 8.8 19.1? 1.9

+ 0.15

62.0 -e 3.0 8.7 -+ 0.5 53.0 + 4.0 22.0 t 3.0 226.0 + 18.0 0.58 -+ 0.06 18.70 ? 0.10

65.3 t 0.0 11.0 5 0.1 54.3 + 0.2 4.15 + 0.15 154.2 -e 0.5 0.24 + 0.01 18.10 % 0.00

11.0 L 2.0 56.9 + 0.9 2 1.0 -+ 2.0 233 0 ? 5 0 0.90 .f 0:20 18.73 t 0.04

19.0 t 1.0 47.0 23.0 206.0 0.80 18.80 -+ 3.0 t 3.0 -e 15.0 +- 0.30 -+ 0.10

10.7 + 0.4 52.3 ? 3.6

52.5 k 6.4 4.4 t 0.8 142.0 + 23.0 0.12 + 0.05 17.90 2 0.10

13.1 + 1.0 197.0 IT 14.0 0.86 2 0.09 18.65 2 0.05

Values lnolccules.

are means

+ SE (no. of preparations

in parentheses).

Unsaturation

index

is defined

as the number

of double

bonds

per 100 fatty

acid

moles percent of total identified fatty acids. Quantitatively, the most abundant fatty acids were palmitate ( 16:0>, stearate (l&O), oleate [ 18: l(n-9>], linoleate [18: 2( n-6)], arachidonate [20:4(n-6)], and docosahexaenoate [22:6(n-3)], with 20:5(n-3) and 22:5(n-3) also making quantitatively significant contributions in sheep. The amount of saturated fatty acids remained constant over the range of body masses studied, with palmitate (16:0) and stearate (l&O) making the major contributions. Correlations were sought between all data in Tables 2 and 3 and body mass to establish whether fatty acid compositional differences were related to body mass. Significant correlations (P < 0.05) were found for the following: unsaturation index (exponent -0.04, Fig. 4A), monounsaturated fatty acid content (exponent 0.07, Fig. 4B), 16:0 content (exponent -0.06, Fig. 5A), 18:O content (exponent 0.05, Fig. 5B), and 22:6(n-3) content (exponent -0.28, Fig. 5C). Less significant correlations (P < 0.1, not shown) were found for 18:l(n-9) (exponent 0.09, r = 0.662), 18:3(n-3) (exponent 0.30, r = 0.725), 20:3(n-6) (exponent -0.12, r = -0.619), and 20:4(n-6) (exponent -0.09, r = Table 3. Fatty acid composition
Mouse
13ody m;lss,

-0.596). No other individual set of data listed in either Table 2 or 3 correlated with body mass. Some collective parameters were also considered for correlation with body mass. The total content of 22carbon fatty acids correlated with body mass with low significance (exponent -0.21, r = -0.624, n = 9, P < 0.1). The ratio of 20:4(n-6)/18:2(n-6) (Table 2>, sometimes used as an indicator of A5- and A6-desaturase activity, showed a nonsignificant correlation with body mass (exponent -0.14, r = -0.557, n = 9; NS). However, 22:6(n-3)/22:5(n-3), which might be taken as a measure of A4-desaturase activity, correlated highly significantly with body mass (Fig. 50; exponent - 0.33). The fatty acid data for rat liver mitochondria in Tables 2 and 3 are similar to some of the previously reported fatty acid compositions for the rat (5, 10, 21, 33, 38); however, there are some differences within any given composition profile. In summary, unsaturation index; the content of 16:0, 18:0, 18: l(n-9>, 18:3(n-3), 20:3(n-6), 20:4(n-6), and 22:6(n-3); monounsaturated fatty acids; and total 22isolated
New Zealand Rabbit (1) 33.0 12.00 0.40 1.00 23.00 0.80 17.40 12.00 2.20 0.70 0.70 9.00 4.40 0.20 8.00 0.50 8.00

Downloaded from ajpregu.physiology.org on May 18, 2009

ofphospholipids
Rat 0.25 11.90 0.60 0.43 25.00 1.90 3.70 13.20 0.00 1.78 0.40 20.30 1.80 0.03 1.30 0.00 13.00 + 0.00 (9) -+ 0.50 + 0.04 -+ 0.08 + 1.00 t 0.10 -c 0.60 t 0.40 + 0.00 + 0.07 + 0.10 + 0.20 t 0.30 + 0.03 lr 0.20 k 0.00 + 2.00

of liver mitochondria
Ferret 1.00 13.00 0.60 0.37 17.00 2.80 8.00 21.00 0.13 1.40 1.00 18.00 1.20 1.85 3.70 0.30 8.00 2 0.20 + 1.00 + 0.20 f 0.04 2 1.00 + 0.20 + 1.00 t- 1.00 + 0.08 -e 0.60 t 0.20 t 3.00 + 0.50 -e 0.05 Ifr 0.90 k 0.00 + 2.00 (3) Dutch 2.35 12.00 0.30 0.60 22.00 1.20 9.10 39.10 0.90 0.50 0.45 9.30 0.20 0.40 0.85 0.50 2.15 Rabbit 1- 0.15 + 0.60 + 0.00 +- 0.00 -t 0.60 -+ 0.10 t 0.40 + 0.50 -e 0.10 -+ 0.00 + 0.05 t 0.20 -e 0.00 2 0.10 t 0.05 -e 0.00 + 0.15 (2)

from different
Sheep -t 0.0 (3) -+ 2.00 t 0.07 + 0.10 ? 3.00 t 0.07 + 0.60 * 1.00 + 0.20 + 0.10 -+ 0.20 -+ 3.00 t 0.90 2 0.20 + 1.00 t 0.00 + 1.00

mammals
Pig 40.0 10.10 0.50 0.45 26.00 1.40 8.20 19.80 0.45 1.00 0.50 17.00 2.50 0.35 2.80 0.05 7.00 f 5.0 (2) + 0.70 -+ 0.10 2 0.05 +- 3.00 + 0.10 -+ 0.00 + 0.40 + 0.05 + 0.00 + 0.00 + 1.00 + 0.30 + 0.05 + 0.00 k 0.05 + 0.40 Horse 340 12.00 1.30 0.45 22.30 1.70 14.00 34.00 2.20 0.45 0.40 4.00 0.75 0.00 0.40 0.00 0.40 (2) ? 2.00 + 0.70 + 0.15 + 0.90 ? 0.60 + 2.00 + 4.00 + 1.30 k 0.05 + 0.20 2 1.00 2 0.05 t 0.00 2 0.00 t 0.00 -+ 0.20

Hamster 0.10 24.00 0.53 0.60 14.00 1.20 6.20 19.00 0.23 0.70 0.53 11.00 1.30 0.03 1.00 0.30 20.00 + 0.00 (3) + 2.00 t- 0.04 -+ 0.06 f 2.00 + 0.07 5 0.10 + 1.00 + 0.04 t- 0.07 -+ 0.04 -+ 0.50 ? 0.20 t 0.03 + 0.20 2 0.04 k 2.00

kg

16:O 16: l(n-7) 17:o 18:O

18: lfn-7)
lH:l(n-9) 18:2(11-6) 18::1( 20::3( 2o::u I>-:$) n-6) 11-9) n-6) n--:3 1

0.02 19.00 0.90 0.25 14.00 2.00 8.00 16.00

0.00 2.00

t 0.00 (3) -+ 1.00 + 0.30 L 0.05 IIt 2.00 2 0.40 -+-2.00 -+ 2.00
f + 0.00

20:4(
20:5(

0.50 14.00
1.60

0.10 I+ 0.07 -+ 2.00

+ 0.50

22:4(11-G)
22:N 22:5(11-6) 22:6(r-3) II-Z3 1

0.00 t 0.00 0 65 ! 0 OFi

o:o;, 13.00 + + 0:oo 5.00

5.06 11.1 0.5 0.6 21.0 1.1 7.9 33.5 1.3 0.4 0.3 11.7 0.3 0.7 1.0 0.9 3.1

Values

are means

k SE (no. of preparations

in parentheses)

in CTomoles. Only

identified

fatty

acids

are given.

R1556

MITOCHONDRIAL

PROTON

LEAK

AND

BODY

MASS

y = 200.5x-0*0401

r =- 0.705,

p = 0.034

= 10.78x0.070 , r = 0.685, p = 0.042

Fig. 4. General properties of fatty acids from liver mitochondrial phospholipids as a function of body mass. A: unsaturation index of fatty acids; B: monounsaturated fatty acids (mol% of total). Data from Table 2. Lines were fitted using linear regression. I
0.01 0.1

I
1

I
10

I
loo loo0

s 0.01 0.1

10

100

1000

body mass (kg)

body mass (kg)

carbon fatty acids all correlated with body mass to some degree. Proton leak per unit surface area and its relationship to body mass. The results above show with isolated liver mitochondria that proton leak per milligram protein, membrane surface area, and phospholipid fatty acid composition all correlate with body mass. To examine if surface area was important in determining the apparent proton permeability, we recalculated the proton leak rate at 170 mV on the basis of microliter matrix volume, milligram phospholipid, and square centimeter membrane surface area using the data in Table 1. The results are shown in Fig. 1. Figure 1B shows that proton leak per microliter matrix volume correlated significantly with body mass with exponent -0.133 t 0.037 (SE). Thus there is a correlation between body mass and proton leak expressed on the basis of amount (mg protein or matrix volume) of mitochondria. However, proton leak expressed per milligram phospholipid (Fig. 1C) or per square centimeter surface area (Fig. lo>, measures of the amount of membrane present, showed nonsignificant correlation with body mass, with exponents of only -0.054 t 0.045 (SE) and -0.027 ? 0.033 (SE).

Comparison of the exponents in Fig. 1 shows that if we assume a constant proton leak rate per unit surface area at 170 mV, we can explain most of the correlation of proton leak per milligram protein with body mass in mammals. From the mean slopes in Fig. 1,A and B, and Fig. 1, C and D, we calculate that allometric changes in surface area could account for 69.1 t 22.5% (SE) of the observed changes in proton leak rate per milligram mitochondrial protein. Proton leak per unit surface area and its relationship to phospholipid fatty acid content. Although there were significant correlations (P < 0.05) between proton leak per milligram protein or per microliter matrix volume and various compositional parameters such as unsaturation index (not shown), these may have been due to the related changes in surface area and not to changes in composition itself. To examine if fatty acid composition was important in determining the proton leak rate differences in liver mitochondria from mammals of different body mass, we looked for correlations between proton leak per unit surface area and the fatty acid composition of the mitochondrial phospholipids. The data in Tables 2 and 3 were plotted against proton leak

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A 3 100 2 :
5 33 2; 1(-jy = 14.17x-0*063, r = -0.715, p = 0.030

B
y = 19.06x0*053 , r = 0.710, p = 0.032

l a

+a

85 G2: b Fig. 5. Fatty acids of mitochondrial phospholipids as a function of body mass. A: 16:0 (mol% of total); B: 18:0 (mol% of total); C: 22:6(n-3) (mol% of total); D: ratio of 22:6(n-3) content to 22:5(n-3) content. Data calculated from Table 3. Lines were fitted using linear regression. t.Yi 2 w loo1 . oll . ; i. lh 1ooo

t----i

0.01

0.1

10

100

1000

y = 6.978x-os276,

r = -0.728, p = 0.026

. s
I I I

0.01

0.1

100

1000

0.01

0.1

100

1000

bodi mas%g)

bodi mas?(k@

MITOCHONDRIAL

PROTON

LEAK

AND

BODY

MASS

R1557

rate per milligram phospholipid and proton leak rate per square centimeter of inner membrane. Significant correlation (P = 0.004) was observed between 20:3(n-6) content and proton leak per milligram phospholipid (Fig. 6A), and there was weak correlation (P = 0.07) between unsaturation index and proton leak per milligram phospholipid (Fig. 6B). No other significant correlations were found. However, there was a tendency for proton leak expressed in either way to increase with increases in the n-3 polyunsaturated fatty acid (PUFA) content and to decrease with increases in the 18:2(n-6) content (not shown). Thus from this type of analysis there seems to be only a minor role for differences in phospholipid fatty acid composition in determining the differences in proton leakiness between liver mitochondria from mammals of different body mass. Mitochondrial phospholipid fatty acid content and proton leak rates in general. To see whether the trends connecting proton leak rate to phospholipid fatty acid content observed above were more apparent with a wider range of leak rates and compositions, we combined literature data for liver mitochondrial phospholipid fatty acid content and proton leak rates from thyroid status and phylogenetic studies with our data. Fatty acid content values for mitochondrial comparisons were normalized against the corresponding control rat values from the same study. Values can be found in the literature as follows: liver mitochondrial phospholipid fatty acid values from hypothyroid (10,21,38) and hyperthyroid rats (33) and lizards (5); proton leak rate per milligram protein for hypothyroid (18) and hyperthyroid rats (20) and lizards (5); total mitochondrial phospholipid-to-protein ratio for hypothyroid and hyperthyroid rats (7) and lizards [O.l, i.e., the same as rat, because surface area per mg protein is similar in the two species (5)]. Figure 7 shows that there were significant positive correlations (P < 0.04) of unsaturation index (Fig. 7A) and percent n-3 PUFA content (Fig. 7B) with proton leak per milligram phospholipid at 170 mV for this data set. There was a less significant negative correlation (P = 0.09) for 18:2(n-6) content. No equivalent data for 20:3(n-6) content could be found for hypothyroid or hyperthyroid rat. However, when normalized 20:3(n-6) content for lizard was combined with the data in the present study, there was no significant correlation with

proton leak. There were similar trends, but no significant correlations, between proton leak per square centimeter inner membrane and fatty acid composition for this larger data set (using surface area data from Refs. 5 and 7).
DISCUSSION

We have previously shown that proton leak rate significantly correlates with body mass in mitochondria isolated from liver (exponent -0.14, r = (0.59)1/2 = -0.768, n = 8, P < 0.05) over a body mass range of 7,500-fold (28). The range in body mass for the present study was 17,000-fold and the proton leak rates per milligram protein at 170 mV were significantly related to body mass, with an exponent of -0.13 (Fig. 1A). Hence, with an increase in body mass of 17,000-fold, there is a 3.5-fold decrease in proton leak rate. Thus the proton leak rates per milligram protein measured in this study correlate with body mass as before (28). In contrast to proton leak rates, the maximal electron transport chain activity per milligram mitochondrial protein (Table 1) does not correlate with body mass. This result is consistent with an allometric study of isolated skeletal muscle mitochondria reported by Taylor (36). Predictably, therefore, the respiratory control ratio increases with increasing body mass (Fig. 2). In general terms there is a 3.2-fold increase in respiratory control ratio of liver mitochondria with a 17,000-fold increase in body mass. Because the procedure for isolating liver mitochondria was optimized for rat (9), although mitochondria from larger animals show even better coupling, we can be confident that damage during isolation did not affect the integrity of the mitochondria isolated from the livers of other mammals. At the same driving force of 170 mV, the observed differences in the proton leak rates per milligram protein could be caused by 1) the amount of mitochondrial inner membrane surface area available per milligram protein for protons to cross or 2) differences in some aspect of the composition of the mitochondrial inner membrane that affect its leakiness. Importantly, we know that the lower proton leak rate per milligram protein in horse compared with mouse is not due to an increased amount of nonmitochondrial protein contamination of the liver mitochondria (28).

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4000

1
140 160 180

r= 0.629, p = 0.070

Fig. 6. Proton leak rate per mg phospholipid at 170 mV as a function of fatty acid content of mitochondrial phospholipids. A: proton leak rate per mg phospholipid as a function of 20:3(n-6) content; B: proton leak rate per mg phospholipid as a function of unsaturation index. Data calculated from Tables l-3. Lines were fitted using linear regression.
240

-6

20:3n6 content (moles

% total

fatty

a cids)

5E

200

220

unsaturation

index

R1558

MITOCHONDRIAL

PROTON

LEAK

AND

BODY

MASS

r = 0.565, p = 0.035
8 0

I loo

150

200

250

normalized

unsaturation

index

r= 0.606, p= 0.037 I

normalized %n3 content (moles % total fatty acids)

r = -0.471, p = 0.089

kc4 0 E
23

00
10

20

30

40

50

normalized 18:2n6 content (moles % total fatty acids)

Fig. 7. Proton leak rate per mg phospholipid at 170 mV of a wider range of mitochondria as a function of the fatty acid content of mitochondrial phospholipids. A: proton leak rate per mg phospholipid as a function of unsaturation index; B: proton leak rate per mg phospholipid as a function of %n-3 polyunsaturated fatty acid content; C: proton leak rate per mg phospholipid as a function of 18:2(n-6) content. Data from Fig. 6 for mammals of different body mass; from Ref. 5 for a lizard species; from Refs. 7, 10, 18, 21, and 37 for hypothyroid rats; and from Refs. 7, 19, and 32 for hyperthyroid rats. Literature data were normalized by expressing it as a fraction of literature control rat value then multiplying by the rat value in Fig. 6. Lines were fitted using linear regression.

Our results show that both mitochondrial membrane surface area (measured either by electron microscopy or by phospholipid content) (Fig. 3) and the fatty acid composition of the membrane phospholipids (Figs. 4 and 5) change as body mass changes. Because proton leak per milligram protein also correlates with body mass (Fig. lA), a demonstration that proton leak correlates with surface area or with composition is not in itself sufficient to establish a causal connection. For example, it cou.ld be that leak depends only on surface area, but that it also correlates with camp osition be-

cause of the simultaneous variation in composition with body mass. To eliminate the effect of surface area, we expressed the leak per surface area (Fig. 1, C and D) and found that there was now no significant correlation between leak and body mass. There was also no significant correlation between leak per surface area and fatty acid composition, except for the unsaturation index and the minor phospholipid fatty acid 20:3(n-6), which showed some correlation with leak per milligram phospholipid (Fig. 6) but not with leak per square centimeter. From the allometric exponents in Fig. 1 we can conclude that differences in surface area account for -70% of the differences in proton leak per milligram protein in mammals of different body mass. If there are effects of fatty acid unsaturation on the proton leakiness of the mitochondrial inner membrane, these are not a major cause of the dependence of proton leak on body mass in mammals, and the scatter in the data makes it impossible to see any such effects unambiguously. The opposite result has been found when accounting for differences in mitochondrial proton leak rate per milligram protein in a mammal-reptile comparison and, to some extent, in studies of the effects of thyroid status in rats. In liver mitochondria isolated from a mammal (rat) and a reptile (bearded dragon lizard; Amphibolurus uitticeps), the proton leak rate per milligram protein at 37C at any given membrane potential was approximately fourfold greater in rat compared with lizard (5). However, there was very little difference in the liver mitochondrial inner membrane surface areas per milligram protein between these two species, so the difference in proton leak rate per milligram protein between the mitochondria must lie with differences in the composition of the inner membrane. Marked differences in phospholipid fatty acid composition were observed (5). In liver mitochondria isolated from hyperthyroid rats compared with hypothyroid rats, only a proportion (2- to 3-fold) of the sevenfold greater mitochondrial proton leak rate per milligram protein could be accounted for by an increase in mitochondrial inner membrane surface area. The remainder of the difference must have been due to inner membrane compositional differences (7,lS); fatty acid compositional differences are known to occur (10, 14, 21, 33, 38). It may be that both surface area and compositional effects occur in nature, but surface area effects dominate in the allometric differences in proton leak and compositional effects dominate in the phylogenetic differences, whereas thyroid effects show an intermediate pattern. When we took allometric, phylogenetic, and thyroid status data together (Fig. 7), we found significant positive correlations of unsaturation index and percent n-3 PUFA content with proton leak per milligram phospholipid at 170 mV and a weaker negative correlation between 18:2(n-6) content and proton leak per milligram phospholipid. These findings agree with previous conclusions that unsaturation index and n-3 PUFA are associated with an increase, and 18:2(n-6) is associated with a decrease, in the proton permeability of mitochondrial membranes (4-7, 21, 27, 31). Studies

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MITOCHONDRIAL

PROTON

LEAK

AND

BODY

MASS

R1559

on plasma membrane fatty acid composition and ion leak rates have produced similar results in terms of n-3 PUFAs. Stillwell et al. (35) have shown that 22:6(n-3) in phosphatidylcholine increases permeability of lipid vesicles and tumor cells. Couture (12) has demonstrated that K+ efflux from liver slices correlates with the sum of 22:5(n-3) plus 22:6(n-3) content of the tissue. A direct correlation of proton leak rate with n-3 PUFA is also consistent with ion permeability models. According to Eyrings model (17), the energy barrier to ion diffusion is greatest at the center of the bilayer. Therefore, the n-3 PUFAs might be expected to be the species of PUFA most likely to increase bilayer permeability. Our results are also consistent with membrane permeability being a direct function of the number of double bonds present in the fatty acids in the phospholipids of the bilayer as discussed by King et al. (24). In this study we have assumed that the fatty acids of the phospholipids in the outer membrane are similar to those of the inner membrane. In liver there are differences in fatty acid composition between outer and inner membranes (11). However, when the molar proportions of fatty acids from the inner membrane are compared with total mitochondrial fatty acids, the results are very similar (Ref. 11 and P. S. Brookes and M. D. Brand, unpublished observation). Hence, the fatty acid composition of whole mitochondria seems, in rat liver at least, to accurately reflect the composition of the inner membrane. In conclusion, our results indicate that the correlation between proton leak rate per milligram protein at 170 mV in isolated liver mitochondria and the body mass of mammals is caused mostly (70%) by differences in mitochondrial surface area. Compositional differences must be responsible for any remaining effect; there is a weak correlation between unsaturation index of phospholipid fatty acids and proton leak per milligram phospholipid, an indicator of mitochondrial bilayer area.
We are grateful to Andrew Grace, Jamie Vandenberg, Hugh Bethel, Nick Gay, Mark Carrington, and Robin Hesketh (Dept. of Biochemistry, University of Cambridge); Nick Rutherford (Dept. of Biochemistry, University of Leeds); Abby Fowden and Marion Silver (Dept. of Physiology, University of Cambridge); and Richard Eastwood, Leslie Hall, George Cremona, Celia Marr, and Bill McGoldrick (Dept. of Veterinary Medicine, University of Cambridge) for tissue samples. We thank Julie Buckingham, Mark Leach, John Richardson, and Pat Pledger (Dept. of Biochemistry, University of Cambridge); Paul Hughes (Dept. of Physiology, University of Cambridge); Wojtech Mantaj (Dept. of Biological Sciences, University of Wollongong); and Richard Eastwood for technical assistance; and David Rolfe, Paul Brookes, and the referees for constructive criticisms of this manuscript. Electron microscopy was by Dan Hill (Dept. of Biochemistry, University of Cambridge). Funding for this work was provided by the Agricultural and Food Research Council (UK) and the Australian Research Council. Address for reprint requests: R. Porter, Dept. of Biochemistry, Trinity College Dublin, Dublin 2, Republic of Ireland. Received 26 January 1996; accepted in final form 14 May 1996.

REFERENCES 1. Ames, B. N. Assay of inorganic uhosnhatases. Methods Enzymol. phosphate, total 8: 11%118,1966. phosphate and

K. The minimal metabolism. In: Energy Metabolism in 2. Blaxter, Animals and Man. Cambridge, UK: Cambridge University Press, 1989, p. 120-146. 3. Brand, M. D. Measurement of mitochondrial protonmotive force. In: Bioenergetics-A Practical Approach, edited by G. C. Brown and C. E. Cooper. Oxford, UK: IRL, 1995, p. 39-62. 4. Brand, M. D., L.-F. Chien, E. F. Ainscow, D. F. S. Rolfe, and R. K. Porter. The causes and functions of mitochondrial proton leak. Biochim. Biophys. Acta 1187: 132-139,1994. 5. Brand, M. D., P. Couture, P. L. Else, K. W. Withers, and A. J. Hulbert. Evolution of energy metabolism: proton permeability of the inner membrane of liver mitochondria is greater in a mammal than in a reptile. Biochem. J. 275: 81-86,199l. 6. Brand, M. D., P. Couture, and A. J. Hulbert. Liposomes from mammalian liver mitochondria are more polyunsaturated and leakier to protons than those from reptiles. Comp. Biochem. Physiol. B Comp. Biochem. 108: 181-188,1994. 7. Brand, M. D., D. Steverding, B. Kadenbach, P. M. Stevenson, and R. P. Hafner. The mechanism of the increase in mitochondrial proton permeability induced by thyroid hormone. Eur. J. Biochem. 206: 775-781,1992. 8. Brody, S. Basal energy and protein metabolism in relation to body weight in mature animals of different species. In: Bioenergetits and Growth. New York: Reinhold, 1945, p. 352-403. 9. Chappell, J. B., and R. G. Hansford. SubcelLular Components: Preparation and Fractionation (2nd ed.), edited by G. D. Birnie. London: Butterworth, 1972, p. 77-91. 10. Clejan, S., P. J. Collip, and V. T. Maddaiah. Hormones and liver mitochondria: influence of growth hormone, thyroxine, testosterone, and insulin on thermotropic effects of respiration and fatty acid composition of membranes. Arch. Biochem. Biophys. 203: 744-752,198O. 11. Colbeau, A., J. Nachbaur, and P. M. Vignais. Enzymic characterization and lipid composition of rat liver subcellular membranes. Biochim. Biophys. Acta 249: 462-492,197l. 12. Couture, P. Body Size, Cell Membranes and Tissue Metabolism in Mammals (PhD thesis). Wollongong, Australia: University of Wollongong, 1993. 13. Couture, P., and A. J. Hulbert. Relationship between body mass, tissue respiration rate, and sodium pump activity in mammalian liver and kidney. Am. J. Physiol. 268 (Regulatory Integrative Comp. Physiol. 37): R641-R650,1995. 14. Daum, G. Lipids of mitochondria. Biochim. Biophys. Acta 822: l-42,1985. 15. Else, P. L., and A. J. Hulbert. An allometric comparison of the mitochondria of mammalian and reptilian tissues: the implications for the evolution of endothermy. J. Comp. Physiol. B Biochem. Syst. Environ. Physiol. 156: 3-11, 1985. 16. Else, P. L., and A. J. Hulbert. Mammals: an allometric study of metabolism at tissue and mitochondrial level. Am. J. PhysioZ. 248 (ReguZatory Integrative Comp. Physiol. 17): R415-R421, 1985. 17. Garlid, K. D., A. D. Beavis, and S. K. Ratkje. On the nature of ion leaks in energy-transducing membranes. Biochim. Biophys. Acta 976: 109-120,1989. 18. Hafner, R. P., C. D. Nobes, A. D. McGown, and M. D. Brand. Altered relationship between proton motive force and respiration rate in non-phosphorylating liver mitochondria isolated from rats of different thyroid status. Eur. J. Biochem. 178: 511-518, 1988. 19. Harper, M.-E., J. S. Ballantyne, M. Leach, and M. D. Brand. Effects of thyroid hormones on oxidative phosphorylation. Biothem. Sot. Dans. 21: 785-792,1993. M.-E., and M. D. Brand. The quantitative contribu20. Harper, tions of mitochondrial proton leak and ATP turnover reactions to the changed respiration rates of hepatocytes from rats of different thyroid status. J. BioZ. Chem. 268: 14850-14860, 1993. 21. Hoch, F. L. Lipids and thyroid hormones. Prog. Lipid Res. 27: 199-270,1988. 22. Horrum, M. A., R. B. Tobin, and R. E. Eklund. Thyroid hormone effects on state IV proton motive force in rat liver mitochondria. MOL. Cell. Endocrinol. 68: 137-141, 1991. A. J., and P. L. Else. Evolution of mammalian 23. Hulbert, endothermic metabolism: mitochondrial activity and changes in

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PROTON

LEAK

AND

BODY

MASS

24.

25. 26. 27.

28

29

30.

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