We investigated the relationship between body size, Na+-K+-ATPase molecular activity, and membrane lipid composition in the kidney of five mammalian and eight avian species ranging from 30-g mice to 280-kg cattle and 13-g zebra finches to 35-kg emus, respectively. Na+-K+-ATPase activity was found to be higher in the smaller species of both groups. In small mammals, the higher Na+-K+-ATPase activity was primarily the result of an increase in the molecular activity (turnover rate) of individual enzymes, whereas in small birds the higher Na+-K+-ATPase activity was the result of an increased enzyme concentration. Phospholipids from both mammals and birds contained a relatively constant percentage of unsaturated fatty acids; however, phospholipids from the smaller species were generally more polyunsaturated, and a complementary significant allometric increase in monounsaturate content was observed in the larger species. In particular, the relative content of the highly polyunsaturated docosahexaenoic acid [22:6(n-3)] displayed the greatest variation with body mass, scaling with allometric exponents of −0.21 and −0.26 in the mammals and birds, respectively. This allometric variation in fatty acid composition was correlated with Na+-K+-ATPase molecular activity in mammals, whereas in birds molecular activity only correlated with membrane cholesterol content. These relationships are discussed with respect to the metabolic intensity of different-sized animals.
- basal metabolic rate
- fatty acids
the basal metabolic rate (BMR) of vertebrates represents the sum of their metabolic activities under resting conditions. In both mammals and birds, BMR is allometrically related to body mass (5, 30, 32). Compared with their larger counterparts, small mammals and birds have a high metabolic rate when expressed on a mass-specific basis. The relationship is approximately proportional to mass−0.25 (30), which translates to a 15% decrease in mass-specific metabolic rate for every doubling in body mass. Although this body size-related variation in metabolic rate has been known for more than a century (41), its mechanistic basis is still unclear.
The internal organs (kidney, splanchnic organs, heart, lungs, and brain) are major contributors to BMR, accounting for up to 70% of energy expenditure, despite being only ∼8% of total body mass (40). Although some tissues (e.g., lungs and skeletal muscle) represent a relatively constant proportion of body mass, other metabolically active tissues (e.g., heart, liver, kidney, and brain) are relatively larger in smaller mammals and birds and therefore contribute to their increased BMR (13, 19, 36). The scaling of BMR, however, is not fully explained by increased organ size in smaller species but also depends on differences in the rate of metabolism of the tissues. Tissue slices (liver, kidney, brain, spleen, and lung) from smaller mammals respire at a higher rate compared with larger mammals (9, 31), with the same allometric trends also observed in isolated hepatocytes of mammals (37) and birds (12).
At the cellular level, BMR is made up of several processes, including synthetic processes and the maintenance of ion gradients across the plasma membrane. One of the most important gradients is the Na+ gradient, which is maintained by the membrane-bound Na+-K+-ATPase (Na+ pump), a ubiquitous enzyme found in all animal cells. In humans and rats, the in vivo activity of the Na+-K+-ATPase is estimated to account for ∼20% of basal metabolism (40); however, its energy use varies among different tissues, accounting for up to 50–70% of metabolism in brain and kidney (7).
The relationship between Na+-K+-ATPase activity and body mass has been previously investigated in kidney and liver slices from mammals (9). In a comparison of five species, ranging from mice to cattle, Na+-K+-ATPase activity (measured as 86Rb+ uptake) was found to be higher in smaller mammals, declining with allometric exponents of −0.13 and −0.14 in the kidney and liver, respectively. This study, however, did not address whether small mammals had increased Na+-K+-ATPase activity as a result of an increased concentration of Na+-K+-ATPase enzymes or whether there was an increased molecular activity (i.e., turnover rate of substrate per enzyme).
Changes in the molecular activity of membrane-bound proteins have been implicated as a major mechanism underpinning differences in metabolism (22, 23). Thus, in the first part of the present study, we investigate whether the increased Na+-K+-ATPase activity previously observed in more metabolically active small mammals (9) is the result of changes in Na+-K+-ATPase concentration, changes in the molecular activity of individual Na+-K+-ATPase enzymes, or a combination of both. Furthermore, to investigate if changes in molecular activity of membrane proteins may be a general mechanism underlying differences in the rate of metabolism between species of different size, Na+-K+-ATPase molecular activity was also determined in the second major group of endotherms, birds.
Previous comparisons of metabolically diverse species have shown that the higher Na+-K+-ATPase molecular activity observed in endotherms compared with ectotherms (15) is causally linked with the fatty acid composition of the surrounding membrane (16, 47). Specifically, higher levels of polyunsaturated fatty acids (PUFA) along with lower levels of monounsaturated fatty acids (MUFA) have been associated with higher Na+-K+-ATPase molecular activity. Membrane fatty acid composition also varies with body mass in tissues of mammals (8, 25), in mammalian mitochondria (38), and in the skeletal muscle (24) and liver mitochondria of birds (4). These allometric comparisons indicate that, in general, small mammals and birds have increased levels of membrane PUFA and decreased levels of MUFA compared with their larger counterparts. In the second part of the current investigation, we specifically determine the relationship between kidney microsomal membrane lipid composition and body mass and examine whether variation in membrane lipid composition may have a role in underpinning any differences in the molecular activity of the Na+-K+-ATPase.
MATERIALS AND METHODS
The mammals and birds examined in the present study were adults of either sex (see Table 1). Mice (Mus musculus) and rats (Rattus norvegicus) were purchased from Gore Hill Research Laboratories (Sydney, NSW, Australia) and housed in the University of Wollongong animal house at 22 ± 2°C with a 12:12-h light-dark photoperiod and ad libitum access to food (rodent pellets) and water. Mice were killed by cervical dislocation, whereas rats were killed by Nembutal overdose (pentobarbitone sodium, 100 mg/kg body mass; ip injection). Tissues from sheep (Ovis aries), pigs (Sus scrofa), and cattle (Bos taurus) were obtained from a local abattoir (Wollondilly, NSW, Australia), where immediately after death the tissues were transported on ice back to the university for immediate use in the experiments. The diet of the sheep, pigs, and cattle before death was unknown.
Emus (Dromaius novaehollandiae) were purchased from Marayong Park Emu Farm (Falls Creek, NSW, Australia). Zebra finches (Taeniopygia guttata), ducks (Anas platyrhynchos), and geese (Anser anser) were purchased from local pet shops or at the Narellan Aviary Bird Auction (NSW, Australia). Feral pigeons (rock dove, Columba livia) were obtained from a local pigeon breeder (T. Cooper, Corrimal, NSW, Australia). House sparrows (Passer domesticus), starlings (Sturnus vulgaris), and pied currawongs (Strepera graculina) were free-living animals trapped 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 body mass ip, except in the case of the emus where the injection was ij).
Body mass and kidney mass of the mice, rats, and all the bird species were obtained immediately after death. For sheep and cattle, carcass weights were used to calculate the mass of the whole mammal, assuming carcass weight was 55% of total body mass as is routinely used commercially (9). The pig carcass weight included the skin, and, because this organ accounts for 15–20% of total body mass, carcass weight was assumed to be 70% of total body mass. Kidney mass for the sheep, pigs, and cattle was obtained immediately before the commencement of experimental assays. All procedures were performed in accordance with the National Health and Medical Research Council Guidelines for Animal Research in Australia and were approved by the Animal Experimentation Ethics committee of the University of Wollongong.
Ouabain was purchased from ICN Biomedicals, [3H]ouabain (30.0 Ci/mmol, 37 MBq, 96.2% purity) from Amersham Pharmacia Biotech (NSW, Australia), scintillation cocktail (Ready Safe) from Beckman (NSW, Australia), tissue solubilizer (Soluene-350) from Packard Biosciences (VIC, Australia), and Na2ATP (special quality) from Boehringer Mannheim (Mannheim, Germany). All other chemicals and reagents were of analytical grade and were purchased from either Merck (Darmstadt, Germany) or Sigma (St. Louis, MO).
The concentration of Na+-K+-ATPase enzymes was determined using the [3H]ouabain binding method described by Else et al. (15). Kidneys were removed from the animals immediately after death, cut into small pieces (2–8 mg), and placed in ice-cold K+-free medium that closely resembled the ionic composition of mammalian and avian plasma (see below). Mixed sections of cortex and medulla were used for all measurements. Mammalian K+-free medium was based on a solution previously used (15) and included (in mM): 125 NaCl, 1.2 MgSO4, 1.2 NaH2PO4, 25 NaHCO3, 1.3 CaCl2, and 5 glucose, pH 7.4. Because we had never previously conducted [3H]ouabain binding in avian kidneys, two different solutions were used to assess [3H]ouabain binding in the birds (in mM): 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 mM 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 mM was chosen for the incubations, since it approximates the documented average plasma K+ concentration for a large number of birds (1, 39). K+-free mediums are generally used in [3H]ouabain binding studies, since K+ is thought to inhibit binding of ouabain to the Na+-K+-ATPase (46); however, Else (11) 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; therefore, their average was used to estimate Na+-K+-ATPase concentration in birds.
Kidney tissue samples were preincubated in ice-cold K+-free medium for 2 × 10-min periods to reduce tissue K+. The samples were then incubated in 2 ml of mammalian or avian [3H]ouabain binding medium containing 1 μCi/ml [3H]ouabain and a final ouabain concentration of 5 × 10−5 M. Parallel incubations containing the same amount of [3H]ouabain and a final concentration of 10−2 M ouabain were also conducted to determine nonspecific binding. Mammalian tissues were assayed at 37°C, whereas 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 samples.
After incubation, the samples were washed five times (8 min/wash) in 3 ml ice-cold K+-free medium to reduce 3H activity associated with nonspecific sites, as previously characterized (11, 15). After the wash procedure, samples were blotted lightly, weighed ( ± 0.01 mg), and placed in 200 μl tissue solubilizer (Soluene-350) overnight. Readysafe scintillation cocktail (2 ml) was added to each vial, and 3H activity was counted on a Wallac 1409 Liquid Scintillation Counter (Turku, Finland) with DPM 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 after subtraction of 3H activity determined in excess ouabain (10−2 M), which was deemed nonspecific uptake. [3H]ouabain binding sites per gram of tissue were determined by multiplying the specific uptake by the total ouabain concentration in the medium. Na+-K+-ATPase concentration was calculated assuming a 1:1 stoichiometry between Na+-K+-ATPase and ouabain binding sites and was expressed as picomoles of Na+-K+-ATPase per gram of kidney wet weight.
Determination of Na+-K+-ATPase activity.
Na+-K+-ATPase activity was determined in kidney homogenates using a modified method of that described by Esmann and Skou (17). Dilute homogenates were prepared (2% wt/vol) in ice-cold 250 mM sucrose, 5 mM EDTA, and 20 mM imidazole (pH 7.4) using a glass-glass homogenizer. A mild detergent treatment was applied to the samples before 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) 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 mM: 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 Na+-K+-ATPase. Enzyme activity was initiated by the addition of 3 mM ATP and allowed to proceed for 5 min. The reaction was terminated by the addition of an equal volume of perchloric acid (0.8 M) at 4°C. Pi was determined as previously described (11). Maximal Na+-K+-ATPase activity was calculated as the difference in Pi liberated (from ATP) in the presence and absence of 1 mM ouabain (minus and plus KCl, respectively). Experiments were conducted in duplicate or better.
Molecular activity is defined as the maximal rate of substrate turnover by a protein and, for the Na+-K+-ATPase, was derived by dividing maximal Na+-K+-ATPase activity (expressed as pmol Pi·mg wet wt−1·min−1) by the Na+-K+-ATPase concentration (in pmol/mg wet wt) for the same preparation. The net result was expressed as the number of ATP molecules hydrolyzed by each Na+-K+-ATPase per minute.
Preparation of microsomal membranes.
All lipid measurements were conducted using microsomal membranes, prepared from kidney homogenates (10% in 250 mM sucrose, 20 mM imidazole, 1 mM EDTA; pH 7.4) that were centrifuged at 3,000 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 mM imidazole and 2 mM EDTA (pH 7.5). One microsomal fraction was prepared per animal, except in the case of the zebra finch and sparrow, where a pooled microsomal fraction was prepared from four birds. Thus, although there was only n = 1 for the lipid measurements in these species, the sample was an average for all birds used and was considered a representative sample of the species. The protein content of microsomal preparations (and tissue homogenates) was determined by the Lowry method, using BSA as the standard.
Analysis of membrane lipid composition.
All solvents used in the lipid extractions were of ultrapure grade. Total lipid was extracted from the microsomal preparation by standard methods (18) using chloroform-methanol (2:1, vol/vol) containing butylated hydroxytoluene (0.01% wt/vol) as an antioxidant. Phospholipids were separated from neutral lipids by solid-phase extraction on Strata SPE SI-2 Silica columns (Phenomenex, NSW, Australia). Fatty acid composition of the phospholipid fraction was determined as described in detail elsewhere (35). Briefly, phospholipid fractions were transmethylated with 14% (wt/vol) boron trifluoride in methanol, and fatty acid methyl esters were separated by gas-liquid chromatography on a Hewlett-Packard 5890 Series II gas chromatograph (Hewlett-Packard, Palo Alto, CA) with a fused silica capillary column. Individual fatty acids were identified by comparing each peak's retention time with those of external standards. The cholesterol content of microsomal preparations was determined by enzymatic assay (Sigma Chemicals). Analysis of phospholipid content was via a phosphorus assay, as described by Mills et al. (33).
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 squares method) of log-transformed values using JMP 4.0.1 software (SAS Institute). Linear correlation coefficients comparing lipid parameters and molecular activity were determined using JMP 4.0.1 software. Figures 1–6 were produced using KaleidaGraph 3.51 software (Synergy Software). Allometric and linear 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 results are reported as means ± SE.
The body mass and mass-specific BMR of the mammals and birds examined in the current study are presented in Table 1. The species were chosen to span as wide a range of body mass as practical, on the basis of availability. Mass-specific BMR was lower in the larger species, with mammalian BMR (kcal·g−1·day−1) = 0.47 × mass (g)−0.27 (P < 0.001) and avian BMR (kcal·g−1·day−1) = 1.06 × mass (g)−0.37 (P < 0.001). The allometric exponent describing mammalian BMR in the current study is close to the value of −0.25 generally found to describe BMR in mammals (30, 36), whereas the allometric exponent observed in the birds (−0.37) is very steep and is the result of the fact that the four smallest birds were passerines, which generally possess higher rates of basal metabolism than nonpasserines, which comprised the four larger species (32).
The kidney represented a smaller percentage of total body mass as the size of the species increased (Table 1). In mammals, the kidney averaged 1.5% of body mass in mice compared with 0.2% in cattle, whereas in birds, kidneys averaged 1.0% of body mass in zebra finches compared with 0.3% in emus. Protein concentration was relatively constant in both groups, averaging 191 ± 8 mg/g wet wt in mammalian kidneys and 176 ± 5 mg/g wet wt in avian kidneys (Table 1).
Na+-K+-ATPase activity values determined in the mammals and birds were examined relative to body mass (Fig. 1), and there was a significant allometric decline in Na+-K+-ATPase activity in the larger birds (exponent −0.16, P < 0.01), with a similar, although not quite significant, trend observed in mammals (exponent −0.07, P = 0.07). Body mass explained 85% of the variability of Na+-K+-ATPase activity in bird kidneys, and for every doubling in body mass there would be a 10.4% decrease in Na+-K+-ATPase activity. Na+-K+-ATPase concentration was relatively constant in the mammalian species, averaging 5,130 pmol/g wet wt (Fig. 2). In contrast to this, Na+-K+-ATPase concentration showed a significant allometric decline (exponent −0.12, P < 0.01) in the larger birds (Fig. 2). Molecular activity values varied approximately threefold in the mammals (8,000–24,000 ATP/min), whereas bird molecular activities showed more modest variation, with values ranging from 5,600 to 11,000 ATP/min. The molecular activity values for birds were determined at 40°C and are thus ∼25% higher than if measured at the same temperature (37°C) as those for the mammals (assuming a thermal quotient, Q10, of 2.0). When molecular activity was considered relative to body mass (Fig. 3), there was a significant decrease (exponent −0.10, P = 0.04) in the mammals, with body mass explaining 85% of the variability. No allometric relationship was observed in bird kidneys, with body mass only explaining 15% of the variability seen in the molecular activity values (Fig. 3).
The fatty acid profile of kidney microsomal phospholipids from mammals and birds are presented in Tables 2 and 3, respectively. In both mammals and birds, the most abundant fatty acids were palmitate (16:0), stearate (18:0), oleate [18:1(n-9)], linoleate [18:2(n-6)], arachidonate [20:4(n-6)], and docosahexaenoate [22:6(n-3)]. When fatty acid parameters were considered with respect to body mass, unsaturation index showed an allometric decline with body mass in the mammals (exponent −0.03, P = 0.06, Fig. 4A), despite there being a relatively constant percentage (76%) of unsaturated fatty acid chains in all species. MUFA content showed a significant allometric increase (exponent 0.10, P = 0.02) in the larger mammals (Fig. 4B), with body mass explaining 88% of the variability in this parameter. This relationship largely reflected changes in the most prominent MUFA, 18:1(n-9) (exponent 0.12, P < 0.01); however, the n-7 MUFA also increased with body size (exponent 0.04, P = 0.05). Total PUFA content was negatively correlated with body mass (exponent −0.02, P = 0.03, Fig. 4C), as was the percentage of long-chain C-20 + C-22 PUFA (exponent −0.04, P = 0.01). The content of the two longest-chain fatty acids in the n-6 and n-3 pathways, namely 22:5(n-6) and 22:6(n-3), both declined with increasing body size, although neither relationship quite reached significance (P = 0.09 and P = 0.08, respectively). It is worth noting that, although the allometric relationship for 22:6(n-3) was not quite significant, it showed the greatest variation of any individual fatty acid (exponent −0.21, Fig. 4D).
In bird microsomal phospholipids, there was no significant body size-related variation observed in the unsaturation index (Fig. 4A), the percentage of total unsaturates, or the percentage of total polyunsaturates (Fig. 4C). There was, however, a significant allometric increase in MUFA content in the larger birds (exponent 0.07, P < 0.01, Fig. 4B), which was similar to the trend observed in mammals and similarly appeared to be largely driven by a significant increase in 18:1(n-9) content (exponent 0.10, P < 0.01). The percentage of n-6 PUFA was essentially constant in bird kidneys, averaging 52.7%, whereas the n-3 PUFA content showed a nonsignificant but substantial decline with body size, resulting in a significant allometric elevation in the n-6-to-n-3 ratio (exponent 0.19, P = 0.05). Of the individual fatty acids, 16:0 showed a significant allometric decrease (exponent −0.07, P = 0.03) in the larger birds, whereas the content of 22:6(n-3) again showed a significant and substantial decline with body size (exponent −0.26, P < 0.01, Fig. 4D).
The other membrane lipid components examined were the cholesterol and phospholipid content. The mammalian species showed little variation in cholesterol and phospholipid content, and as a result, a fairly constant molar ratio of cholesterol/phospholipid was observed (Table 2). In the birds, cholesterol and phospholipid content tended to be higher in the smaller species (Table 3), and, when considered with respect to body mass, there was a significant allometric decrease observed in phospholipid content in the larger birds (exponent −0.05, P < 0.05). The molar ratio of cholesterol/phospholipid in birds was similar to the mammalian ratios and displayed no significant body size-related variation. From the cholesterol/phospholipid ratios, it can be calculated that in kidney microsomes from mammals and birds, there are about two to three phospholipids per molecule of cholesterol.
To assess whether membrane lipids may have been influencing the molecular activity of the Na+-K+-ATPase in mammals and birds, linear correlation coefficients were determined between all individual lipid parameters and Na+-K+-ATPase molecular activity values. Significant positive correlations were observed between Na+-K+-ATPase molecular activity in mammalian kidney and the unsaturation index (P < 0.02, Fig. 5A), the total percentage of PUFA (P < 0.05, Fig. 5B), the percentage of 22:6(n-3) (P < 0.05, Fig. 5C), average chain length (P < 0.01), and the percentage of C-20 + C-22 PUFA (P < 0.02). In birds, there was no correlation observed between Na+-K+-ATPase molecular activity and any fatty acid parameter; the molar ratio of cholesterol/phospholipid did, however, correlate negatively with molecular activity (P < 0.05, Fig. 6).
In the present study, we have investigated mechanisms underlying differences in the metabolic rate of mammals and birds of different body size. In both endothermic groups, kidney Na+-K+-ATPase activity was higher in smaller species and displayed a significant allometric decline with body mass (Fig. 1). These results are consistent with those reported in mammalian kidney slices by Couture and Hulbert (9) and indicate that the activity of the Na+-K+-ATPase reflects differences in mass-specific metabolism. One of the goals of this 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 Na+-K+-ATPase enzymes, an increased molecular activity, or a combination of both. The mammals and birds differed in this respect, each using contrasting strategies to achieve higher Na+-K+-ATPase activity in smaller species. In mammals, kidney Na+-K+-ATPase concentration was fairly constant (Fig. 2), and variation in Na+-K+-ATPase activity resulted primarily from significant allometric changes in the molecular activity of the Na+-K+-ATPase (Fig. 3). In contrast, alterations in Na+-K+-ATPase activity in birds were mainly through changes in enzyme concentration (Fig. 2), with less variation observed in molecular activity values (Fig. 3).
The molecular activity values determined in the mammals in the current study are comparable to those previously reported for mammalian kidney (2, 15, 28). Na+-K+-ATPase measurements in birds are more scarce and have primarily concentrated on salt glands; however, the molecular activity of goose kidney (at 37°C) has been reported to be 11,100 ATP/min (2), which is greater than the molecular activity (≃7,900 ATP/min) determined at 40°C for goose kidney in the current study. When comparing molecular activity values from the literature, one problem that is encountered is that the values have been determined on a variety of preparations, including homogenates, microsomes, and purified enzymes, which have been prepared from different sections of the kidney (cortex/medulla). Also, the molecular activities have been determined using various measurement techniques for both enzyme activity and enzyme concentration. A major strength of the current investigation is that all species were assayed using the same treatment conditions and are therefore directly comparable to each other.
One question that arises is whether the molecular activity differences found between the various species are possibly the result of isoform differences. A number of different isoforms exist for both subunits of the Na+-K+-ATPase (29, 44). Expression of these isoforms appears to be tissue specific, with α1 being the primary isoform of the large catalytic subunit found in the kidney of a variety of mammals and birds (20, 44). This isoform is common to all vertebrates and is considered the “housekeeping” form of Na+-K+-ATPase. Although α1 may be the only isoform present in the kidney of all the species from the current study, this isoform does display some subtle kinetic differences in different animals; in particular, α1 has a low affinity for ouabain in tissues from rodents. To ensure that this had no effect on our measures from the mice and rats, we used saturating concentrations of ouabain in both the Na+-K+-ATPase activity assay and [3H]ouabain binding.
The reason for the increased Na+-K+-ATPase activity in small mammals and birds may potentially be a response to increased Na+ and K+ permeability in these species, since elevated metabolic rate has been associated with increased “leakiness” in cell membranes. For example, liver slices and isolated hepatocytes from the bearded dragon lizard have reduced Na+ and K+ permeability compared with the more metabolically active rat (14), and, although passive Na+ permeability of cells from mammals and birds of different body size has not been measured, the increased 86Rb+ uptake rate (an indirect measure of in vivo K+ permeability) previously observed in small mammals (9) suggests that the high Na+-K+-ATPase activity observed in smaller species in the current study may be associated with the maintenance of normal transmembrane ion concentrations.
Activity associated with the Na+-K+-ATPase is one of the major energy-consuming processes in the kidney, accounting for up to 70% of O2 consumption (7). The major function of the Na+-K+-ATPase in the kidney is the maintenance of the electrochemical gradient for Na+, which in turn acts as a driving force for the secondary active transport of nutrients, metabolites, and ions as well as the secretion of organic acids (28). In intact mammalian kidney (rat, rabbit, dog, and pig), the reabsorptive capacity for Na+ has been reported to be 6–24 μmol Na+·g wet wt−1·min−1, with 24–54% of this Na+ reabsorption linked to the Na+-K+-ATPase (27). From these values, in vivo Na+-K+-ATPase activity would be between 0.03 and 0.26 μmol ATP·mg wet wt−1·h−1, which is ∼10% or less than the maximal in vitro values determined in the current study. Thus it appears that the Na+-K+-ATPase is only operating at a low percentage of its maximal capacity under normal conditions.
By combining maximal Na+-K+-ATPase activity with kidney mass, and using an ATP synthesized/O2 consumed (P/O) ratio of 2.0 (40), we were able to estimate the potential maximal daily energy expenditure by kidney Na+-K+-ATPase enzymes (kcal/day). When these values were compared with the BMR values for the different species, they represented ∼20% and 12% of basal energy metabolism in the mammals and birds, respectively. Of interest is the fact that, in both groups, the allometric exponents for Na+-K+-ATPase energy expenditure (0.76 and 0.67) are very close to the BMR exponents of 0.73 and 0.63 seen in mammals and birds, respectively, indicating that maximal kidney Na+-K+-ATPase activity correlates well with basal metabolism and likely constitutes a relatively constant proportion of metabolic activity irrespective of body size and metabolic rate.
A fairly constant level of protein was found in the kidney of mammals and birds of differing body size in the present study (Table 1), which is consistent with previous findings (9). In other comparisons of metabolically diverse groups, the high level of metabolism in the kidney of an endothermic mammal was associated with 38% more protein than in an ectothermic reptile with low metabolic activity (21). The rate of protein synthesis in mammals is proportional to BMR, scaling with mass0.76 (34). The rate at which protein synthesis scales in birds is currently unknown.
Phospholipid fatty acid composition has been shown to vary with body mass in mammalian tissues (heart, skeletal muscle, kidney, and liver; see Refs. 8 and 25), in bird skeletal muscle (24), and in liver mitochondria from mammals (38) and birds (4). In the current investigation, we examined microsomal membranes in preference to whole tissue, since phospholipids isolated from microsomes (i.e., plasma membrane, Golgi, and endoplasmic reticulum phospholipids) are more representative of the lipids and fatty acids that would be directly surrounding the Na+-K+-ATPase in the plasma membrane rather than whole tissue phospholipids, which would also contain nuclear and mitochondrial membrane fractions.
Our results show that microsomal phospholipids from small mammals were more unsaturated than those of large mammals (Fig. 4A) and contained a higher proportion of PUFA (Fig. 4C) along with a reduced content of MUFA (Fig. 4B). Of particular note was the dramatic variation in the relative content of docosahexaenoic acid [22:6(n-3)], which varied with an allometric exponent of −0.21, which is equal to that previously observed for whole tissue phospholipids from mammalian kidney (25) and is also close to the exponent describing mass-specific BMR. Overall, the body size-related variations in fatty acid composition observed in the mammalian microsomal phospholipids are similar to those previously observed in whole tissue phospholipids (8, 25), and also mammalian liver mitochondria (38), suggesting that allometric variation in membrane fatty acid composition is a phenomenon that occurs in all subcellular membranes in mammals.
There were not as many significant allometric relationships observed in bird phospholipids; however, there were a number of similar trends to those seen in the mammals. Phospholipids from the smaller birds had a reduced content of MUFA compared with their larger counterparts (Fig. 4B), and a significant and substantial allometric decline was also observed in the relative content of 22:6(n-3) (Fig. 4D). Another fatty acid that decreased significantly with body size was palmitic acid (16:0), which is a trend that was also seen in skeletal muscle phospholipids from these birds (24). Many of the parameters that displayed no significant allometric variation (e.g., unsaturation index, PUFA content) were heavily influenced by the fatty acid profile of the zebra finch, which was anomalous compared with the other birds, and deviated substantially from the regression line (see Fig. 4). In particular, phospholipids from the finches contained low amounts of 22:6(n-3), which is generally the longest and most polyunsaturated fatty acid occurring in membranes and therefore affects a large number of the composite parameters. Despite this unusual composition, the allometric exponent describing the variation of 22:6(n-3) was close to the allometric exponent describing mass-specific BMR in the birds, which is similar to what was observed for the mammals. Collectively, these results indicate that allometric variation in fatty acid composition may be a phenomenon that is a general trait of endotherms, despite mammals and birds having evolved endothermy independently. Furthermore, these results support the notion that membrane fatty acid composition, and in particular 22:6(n-3) content, may be an important “pacemaker” of animal metabolism in general (22, 23).
The exact mechanisms responsible for body size-related variations in fatty acid composition 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. We have recently shown that the main membrane parameter that appears to be affected by the diet is the relative percentage of n-6 and n-3 PUFA (26).
It is unlikely, however, that the allometric variation in fatty acid composition observed in the current study was the result of the fatty acid profile of the diet, since the rats and mice consumed exactly the same diet; however, there were very large differences in their fatty acid profiles (Table 2). The rats were dominated by n-6 PUFA, particularly 20:4(n-6), whereas the mice phospholipids contained high levels of n-3 PUFA and in particular 22:6(n-3). Thus, although diet can influence membrane fatty acid composition, other regulatory mechanisms appear to be important in providing the specific composition required by each animal. First, there are the elongase and desaturase enzyme systems, which produce long-chain n-6 and n-3 PUFA from their respective precursors, 18:2(n-6) and 18:3(n-3). There is also the constant remodeling of the membrane through deacylation-reacylation cycles. Rat liver cells only synthesize four molecular species of phosphatidylcholine and phosphatidylethanolamine de novo; all other species are produced by the combined actions of acyltransferases, transacylases, and phospholipases (42). The role of these regulatory processes in determining the allometric variation in membrane fatty acid composition in mammals and birds requires further investigation.
Possibly the most important fatty acid was 22:6(n-3), the content of which varied dramatically in the membrane and was very strongly related to body mass and also possibly metabolic rate. The synthesis and modification of most fatty acids appear to occur in the endoplasmic reticulum; however, it has been proposed that the synthesis of 22:6(n-3) involves a single β-oxidation of 24:6(n-3) in peroxisomes (43). The terminal fatty acid in the n-6 pathway, 22:5(n-6), is also thought to undergo a similar method of biosynthesis, and, although it was present in the membrane in much lower amounts, it showed a very similar allometric variation to that observed for 22:6(n-3) in both mammals and birds (results not shown). Thus, although it is unclear why the content of 22:6(n-3), and to a lesser extent 22:5(n-6), displays such dramatic allometric variation, it is possible that the unusual and complex synthetic pathway that produces these fatty acids may be a regulatory factor that determines the changes observed with body size.
The relationship between membrane lipid composition and Na+-K+-ATPase molecular activity was determined in both groups, and in the mammalian group, significant positive correlations were observed between molecular activity and unsaturation index, the total percentage of PUFA, and the content of 22:6(n-3) (Fig. 5). Although some of the positive correlation observed for unsaturation index (Fig. 5A) and the percentage of PUFA (Fig. 5B) is related to the content of 22:6(n-3) (Fig. 5C), other fatty acids, such as 20:4(n-6) in rat kidney and 20:5(n-3) and 22:5(n-3) in both the sheep and cow kidney (see Table 2), also had a large influence on unsaturation index and the total percentage of PUFA and thus explain some of the correlation seen with molecular activity. Taken collectively, these findings suggest that Na+-K+-ATPase molecular activity is higher in membranes containing high levels of PUFA. Such a contention is supported by several other studies (16, 45, 47), and it is interesting to note that similar relationships have also been reported between liver mitochondrial membrane PUFA and mitochondrial proton leak (3, 6, 38), which represents another significant contributor to BMR (40). In contrast to the mammals, there was no correlation observed between any fatty acid parameter and Na+-K+-ATPase molecular activity in the birds. This lack of correlation, however, may have been related to variations in other membrane lipid components, since the molar ratio of cholesterol to phospholipid did correlate negatively with molecular activity (Fig. 6), which is consistent with other reports (10).
In conclusion, our results show that mammals and birds use different strategies to achieve allometric changes in kidney Na+-K+-ATPase activity; mammals vary the molecular activity of the Na+-K+-ATPase, whereas birds vary the Na+-K+-ATPase concentration. Both endothermic groups displayed relatively similar body size-related variations in microsomal fatty acid composition, with the content of 22:6(n-3), in particular, showing the greatest variation of any fatty acid. Correlations were found between molecular activity and membrane fatty acid composition in mammals and molecular activity and membrane cholesterol in birds, suggesting that the lipid milieu surrounding membrane-bound enzymes, such as the Na+-K+-ATPase, may be an important factor in determining their activity.
This work was supported by a grant from the Australian Research Council.
We thank Dr. P. Abolhasan and Dr. W. A. Buttemer for assistance.
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