Respiration rate of hepatocytes varies with body mass in birds
1 Department of Biomedical Science, Metabolic Research Centre, University of
Wollongong, Wollongong, NSW 2522, Australia
2 Department of Biological Science, Metabolic Research Centre, University of
Wollongong, Wollongong, NSW 2522, Australia
3 MRC-Dunn Human Nutrition Unit, Hills Road, Cambridge CB2 2XY,
UK
* Author for correspondence (e-mail: pelse{at}uow.edu.au)
Accepted 7 April 2004
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Summary |
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Key words: allometry, body mass, metabolism, proton leak, sodium pumping, bird, hepatocyte
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Introduction |
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Birds have evolved endothermy independently of mammals. In this study we
report an allometric analysis of the respiration rate of hepatocytes isolated
from eight bird species ranging from 13 g zebra finches to 35 kg emus. This
represents a 2800-fold variation in body mass. We estimated, by the use of
inhibitors, the quantitative importance of four subcellular processes to the
in vitro oxygen consumption of these hepatocytes. These processes
are: total mitochondrial production of ATP, mitochondrial ATP production
devoted to sodium pumping, mitochondrial proton leak and non-mitochondrial
oxygen consumption. We also compare our findings for birds with those
previously reported for hepatocytes isolated from different-sized mammals
(Porter and Brand, 1995).
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Materials and methods |
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Emus (Dromaius novaehollandiae Latham) were purchased from
Marayong Park Emu Farm (Falls Creek, NSW, Australia). Zebra finches
(Taeniopygia guttata Vieillot), domestic ducks (Anas
platyrhynchos L.) and domestic geese (Anser anser L.) were
purchased from local pet shops or the Narellan Aviary Bird Auction (NSW,
Australia). Feral pigeons (rock dove Columba livia Gmelin) were from
a pigeon breeder (T. Cooper, Corrimal, NSW, Australia). House sparrows
(Passer domesticus L.), starlings (Sturnus vulgaris L.) and
pied currawongs (Strepera graculina Shaw) were trapped in or near
Wollongong, NSW, Australia. All birds were killed by anaesthetic overdose
(sodium pentobarbitone, 100 mg kg1 body mass;
intraperitoneal, except for emus where injection was intrajugular) within a
few days of purchase or capture. When birds were kept in captivity for a short
period before sacrifice, they were provided with access to water and food
ad libitum (mixed bird seed for finches and sparrows and a commercial
mixture of pellets and seed for the ducks and geese). The diet of birds before
their purchase/capture was unknown. The birds used in this study are the same
birds for which other data has been previously reported (see
Hulbert et al., 2002b;
Brand et al., 2003
).
Fresh liver tissue was rapidly removed from each bird and a piece of liver
from the end of the major liver lobe was removed for hepatocyte preparation;
the remainder was used for isolation of liver mitochondria (see
Brand et al., 2003). In the
case of zebra finches and sparrows, whole liver lobes were used for the
preparation of hepatocytes. For hepatocyte isolation the cut section was
trimmed to reveal the common major artery and cannulated. Blood was cleared
using ice-cold pre-wash medium, containing (in mmol l1) 10
Hepes, 25 NaHCO3, 125 NaCl, 3.4 KCl, 1.1
KH2PO4, 1.2 MgSO4 and 11.1 glucose, 0.2%
bovine serum albumin and 0.001% Phenol Red, pH 7.2, and gassed with 95%
O2, 5% CO2, either directly from the perfusion apparatus
or from a syringe passed into the cannulated artery, with blood and medium
exiting via veins cut across the liver section. Liver cells were
isolated by performing 510 min of pre-wash perfusion (at 25°C) with
no recirculation followed by 2030 min of recirculated collagenase
perfusion medium (composition as pre-wash medium with collagenase added at 1 g
l1 and 1 mmol l1 CaCl2) at a
flow rate of 2 ml min1 (for small bird species) to 30 ml
min1 (for large bird species). The liver capsule of
undamaged perfused sections of the liver was teased away and the loose liver
cells were gently shaken out into pre-wash medium. Isolated cells were
filtered through 250 µm nylon gauze, centrifuged twice for 4 min at 100
g and resuspended in suspension medium (as for pre-wash but
containing 1 mmol l1 CaCl2) and stored on ice.
Viabilities were assessed by Trypan Blue exclusion and varied with the body
mass of the species. Preparations from emus, geese and ducks averaged 92%, 93%
and 97% viability, respectively. Pigeon and currawong preparations both had an
average viability of 89%, whilst hepatocyte preparations from starlings,
sparrows and zebra finches averaged viabilities of 73%, 72% and 80%,
respectively. The microscopic appearance of all isolated cell preparations
showed no obvious blebbing.
Oxygen consumption was measured using a Strathkelvin (Glasgow, UK) oxygen electrode connected to a Strathkelvin 781 oxygen meter with electrode output coupled to a ADI Instruments (Sydney, Australia) PowerLab 400 data acquisition system coupled to a Macintosh powerbook. Oxygen consumption measurements were made between 3940°C, representing an intermediate temperature between the normal body temperature of the passerine and non-passerine species examined. Cells were added to the oxygen electrode (final protein concentrations between 0.52.0 mg ml1) and oxygen consumption measured for 510 min following thermal equilibration. Calculations of oxygen consumption rates were corrected for the viability of that preparation. Following measurement of normal oxygen consumption rate, specific inhibitors were added sequentially to block components of metabolism. Oligomycin, a specific inhibitor of mitochondrial ATP synthase, was added stepwise (at 0.001 mg ml1) until the rate of oxygen consumption failed to decrease in response to further additions (usually at 0.0020.004 mg ml1 final concentration). The decrease in respiration following oligomycin addition was thus used to measure respiration devoted to ATP production. Myxothiazol, a specific inhibitor of mitochondrial oxygen consumption, was added stepwise (at 0.01 mg ml1) until oxygen consumption failed to decrease further in response to further additions (usually at 0.020.03 mg ml1 final concentration). This further decrease following maximal myxothiazol inhibition was used to estimate respiration associated with proton leak (rate of respiration with oligomycin minus rate with oligomycin plus myxothiazol). In a number of preparations KCN was also added as a final inhibitor (to a final concentration of 5 mmol l1) to check the effectiveness of myxothiazol. Oxygen consumption of nonmitochondrial respiration was insensitive to myxothiazol. Sodium-pump-associated respiration was determined separately by incubation in the presence of ouabain (a specific inhibitor of the sodium pump) at 103 mol l1 final concentration. The respiration rates of some hepatocyte preparations from a number of species were measured until all oxygen was depleted from the system. These preparations showed constant rates of respiration for periods of up to 45 min over the full scale of oxygen partial pressures.
Dry mass of cells was determined by the difference in mass (to ±0.01 mg) between Eppendorf tubes with and without cells before and after drying, with correction for incubation solution salts. The drying process involved placing 0.2 ml of cell suspension (or incubation medium) in preweighed (dry) 2.5 ml Eppendorf tubes into an oven at 50°C for 5 days, followed by transfer to a vacuum-sealed desiccator with dry silica gel for 2 weeks at room temperature. Protein content was measured by the Lowry method using bovine serum albumin as a standard. Cell density was measured using a haemocytometer.
All figures were constructed using Kaleidagraph version 3.51 (Synergy Software, Reading, PA, USA) and statistics determined using JMP version 3 (SAS Institute, Cary, NC, USA).
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Results |
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The liver represented a smaller percentage of total body mass as the size
of the species increased. In zebra finches the liver averaged 2.71% of body
mass compared to 1.87% in emus. This trend resulted in an allometric
relationship of liver mass to body mass with an exponent of 0.91
(Fig. 2). which is similar to
the situation in mammals where liver mass is related to the 0.87 power of body
mass (see Hulbert and Else,
2000). The respiration rate of hepatocytes isolated from the
different-sized bird species, expressed relative to hepatocyte dry mass and
hepatocyte protein content, are presented in
Fig. 3. There was a
statistically significant allometric decline in hepatocyte respiration with
increasing body mass when expressed relative to dry mass but not when
expressed relative to hepatocyte protein content. The allometric relationship
between hepatocyte respiration rate (HRR; nmol O2
mg1 dry mass min1) and body mass
Mb (kg) is
HRR=5.27xMb0.10
(P<0.001). The allometric exponent was 0.10 (95% confidence
limits 0.04 to 0.16), which means that the respiration rates of
hepatocytes from birds the size of zebra finches are approximately 2.2 times
those of hepatocytes from emu-sized birds. The allometric relationship for
respiration rate relative to hepatocyte protein content had a slope of
0.03, which was not statistically different from zero because the
amount of hepatocyte protein relative to hepatocyte dry mass varied with
species size. For example, protein represented an average 93.5% of dry mass in
zebra finch hepatocytes but only 89.0% of dry mass in emu hepatocytes.
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Measurement of both the cell density and protein content of each hepatocyte preparation allowed calculation of the protein content of the average hepatocyte for each preparation. This data is presented in Fig. 4 and shows that, on average, hepatocytes from smaller birds contain more protein per liver cell. Although the slope of this allometric relationship is not significantly different from zero, the equation indicates that whereas hepatocytes from zebra finches average 0.33 ng protein cell1, those from emus average 0.19 ng protein cell1. The reason for the variation in cell protein content is not known. Cell size was not measured in the current study and thus it is not known whether this was a factor in the differences. We do not know the identity of the additional non-protein component of cells; however, it may be glycogen, lipid or salts. These findings illustrate the potential danger in using protein as the denominator when comparing various biochemical processes between species.
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A number of subcellular processes were maximally inhibited and the proportion of hepatocyte respiration devoted to the activity was estimated by the reduction in the rate of hepatocyte oxygen consumption following such inhibition. All of these subcellular processes, total mitochondrial ATP production, ATP production to run the sodium pump, mitochondrial proton leak and non-mitochondrial oxygen consumption, showed negative allometric relationships with body mass (Fig. 5). The allometric exponents for these relationships are respectively 0.09, 0.11, 0.07 and 0.15, which are all reasonably similar to and within the 95% confidence limits of the exponent (0.10 + 0.06) for total hepatocyte respiration. This shows that irrespective of body size of the bird species, these processes represent approximately the same proportion of total hepatocyte respiration. The exponents for ATP production and proton leak were not statistically different from zero using a 5% significance level, but would be if a 10% significance level was used. The constant in each of the allometric equations in Fig. 5 is the value of the process in a 1 kg bird. From these values, it can be calculated that, for a 1 kg bird, total mitochondrial ATP production constitutes 54%, mitochondrial proton leak is 21% and non-mitochondrial oxygen consumption is 25% of hepatocyte respiration. The non-mitochondrial value is a composite of oxygen consumption by other subcellular organelles such as peroxisomes and by non-mitochondrial enzyme systems such as the desaturases. The energy consumption by the Na+ pump in liver cells in culture constitutes 45% of total hepatocyte ATP production and 24% of total hepatocyte respiration.
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By combining hepatocyte respiration rate with the total mass of the liver in each bird (and assuming that liver is a constant 20% dry mass in all birds) we have been able to estimate the resting oxygen consumption by the total liver for each bird. Fig. 6 presents an allometric plot of this estimated total liver respiration against body mass for the different-sized bird species measured in the present study. The allometric slope of 0.84 for this relationship suggests that respiration by the liver represents a larger proportion of total respiration in bird species of greater body mass in general or it may reflect a difference between passerines and non-passerines. Using the relationship between BMR of the birds examined here, presented in Fig. 1, we can calculate that in a 13 g zebra finch the liver consumes about 2% of the total oxygen consumed by the bird, whilst in a 35 kg emu the corresponding value is 7%. These values are likely to be underestimates, however, because hepatocytes in culture are presumably only consuming energy for self-maintenance, whilst the whole liver in the bird will be consuming additional energy on behalf of other body cells via the maintenance of blood composition.
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Discussion |
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The hepatocyte respiration rate comparisons are illustrated in Fig. 7A, where it can be seen that the relationship for birds had a lower allometric slope than that for either mammals or ectotherms. Whether the size-related trend in viabilities of the hepatocyte preparations in this study (see Materials and methods) had an influence on the observed allometric relationship is not known. Another unknown factor is the fact that respiration rates in the present study were measured at the common temperature of 3940°C. The smaller species in the present study were passerines, which tend to have higher body temperatures than the non-passerines representing the larger species we have measured here. Thus a Q10 correction of the current respiration data to the normal body temperature of the species will probably increase the rates for the small species and decrease those for the larger species with the consequent effect of increasing the allometric exponent relating hepatocyte respiration rate to body mass in birds.
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The absolute and relative composition of hepatocyte respiration for a large and small species of both birds and mammals are plotted in Fig. 7Bi and ii, respectively. Whereas mass-specific respiration differs with body size in birds and mammals (Fig. 7A), when hepatocyte respiration components are compared in relative terms (Fig. 7Bii) the body size effect is no longer obvious and there is a very similar relative composition of metabolism between small and large birds and mammals, even though the non-mitochondrial component of hepatocyte oxygen consumption may be greater in birds than mammals.
The amount of hepatocyte metabolism devoted to cellular Na+
homeostasis in the different bird species was estimated by measurement of the
reduction in oxygen consumption in the presence of excess ouabain (a specific
inhibitor of the Na+/K+-ATPase). This rate also showed
an allometric decline with increasing body mass (see
Fig. 5). Since, when measured
in this manner, this is not a measure of the total enzyme activity of
Na+/K+-ATPase but is rather a measure of the in
vivo activity of the sodium pump to maintain intracellular Na+
homeostasis, it suggests that the relative `sodium leak' is greater in
hepatocytes from small birds than from large bird species. Measurement of
sodium pump activity in liver and kidney slices of mammals of different body
size has similarly shown an allometric decline with increasing body mass
(Couture and Hulbert,
1995).
The findings of the present study parallel those previously reported for
mammals in many respects. The mechanisms underlying these relationships are
not precisely known but the `membrane pacemaker' theory of metabolism (Hulbert
and Else, 1999,
2000
) proposes that both the
amount and composition of cellular membranes underly such allometric variation
in metabolic activity. Specifically, it proposes that polyunsaturated
membranes are associated with high metabolic activity while more
monounsaturated membranes are associated with lower metabolic activity. In
this respect, birds show the same relationships as previously observed in
mammals. We have previously reported that the muscle phospholipids of the same
birds measured in the present study show these trends in fatty acid
composition (Hulbert et al.,
2002b
) and have also found similar trends in the fatty acid
composition of liver mitochondrial membranes from these birds
(Brand et al., 2003
). These
trends in membrane fatty acid composition also exist in other tissues (A. J.
Hulbert, N. Turner, M. D. Brand and P. L. Else, unpublished findings).
Recently, as described in the Introduction, there has been disagreement in
the scientific literature between two schools of thought concerning the
explanation of the metabolismbody size allometric relationship. One
school has proposed a theory to explain this relationship based on the
mathematics of fractal-like distribution networks (West et al.,
1997,
2002
) whilst the other school
has suggested a multiple-causes model, where the final exponent describing the
metabolism body mass exponent is the sum of multiple contributors to
metabolism and control. This has been called the allometric cascade
explanation (Darveau et al.,
2002
). The data from the present study undoubtably supports the
`allometric cascade' model. We have found a significant allometric
relationship describing the mass-specific respiration rate of bird hepatocytes
that is less than that describing the basal metabolic rate of whole birds. We
have found that the total size of the liver also varies allometrically with
body size in birds and that when liver mass and the hepatocyte respiration
rate are combined they come closer to the BMRbody mass relationship. In
this respect, our findings are similar to those already reported for mammals
(Porter and Brand, 1995
;
Couture and Hulbert, 1995
).
There is sure to be more discussion regarding the causal basis of the
allometric relationship between body size and metabolism.
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Acknowledgments |
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References |
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