University of Cambridge, Cambridge CB2 lQW, United Kingdom. Porter,. Richard. K., and Martin. D. Brand. Cellular oxygen consumption depends on body mass ...
Cellular
oxygen consumption
RICHARD Department
K. PORTER AND MARTIN of Biochemistry, University
depends on body mass D. BRAND of Cambridge,
Porter, Richard K., and Martin D. Brand. Cellular oxygen consumption depends on body mass. Am. J. PhysioZ. 269 (Regulatory Integrative Comp. Physiol. 38): R226-R228, P995.-Hepatocytes were isolated from nine species of mammal of different body mass (and standard metabolic rate). The cells were incubated under identical conditions and oxygen consumption measured. The rate of oxygen consumption (per unit mass of cells) scaled with body mass with exponent - 0.18. In general, there was a 5.5fold decrease in oxygen consumption rate with a 12,500-fold increase in body mass. The decrease in oxygen consumption rate was not due to an increase in cell volume with increasing body mass but to a decrease in intrinsic metabolic activity of the cells. This novel finding confirms and explains the decrease in oxygen consumption rate measured in tissue slices from larger mammals by H. A. Krebs (Biochim. Biophys. Acta 4: 249-269, 1950) and recently by P. Couture and A. J. Hulbert [Am. J. Physiol. 268 (Regulatory Integrative Comp. Physiol. 37): R641-R650,1995]. allometry;
standard
metabolic
rate; hepatocytes;
mammals
ofananimalisdefinedas the rate of heat production under conditions that minimize known extra requirements for energy, i.e., in animals that are resting, calm, postabsorptive, and at a thermoneutral temperature (1, 8). In mammals, standard metabolic rate can conveniently be measured as oxygen consumption rate (11). This oxygen consumption in the resting mammal is used 1) to provide energy for mechanical work (e.g., pressure/volume work of the heart and lungs), osmotic work (e.g., excretory work by the kidney), etc., and 2) to maintain, at steady state, low entropy structures, such as proteins and ion gradients. The relationship between standard metabolic rate [heat produced per unit time (E/t)] and body mass (A!) in eutherian mammals is given by the equation E/t = aM”.75, where a is the elevation constant and has a value of 293 when units of kilojoules per day are used (1, 8). Thus the 250-kg horses in our study have a metabolic rate - 1,200 times that of the 20-g mice (Table 1). Similarly, when metabolic rate is expressed per unit body mass, resting oxygen consumption is proportional to M-o.25, and the 20-g mice have 10.5 times the mass specific metabolic rate of the 250-kg horses. The liver is a significant contributor to standard metabolic rate, accounting for - 20% of standard metabolic rate in the rat (6). Differences in standard metabolic rate between mammals of differing body mass are partially due to differences in the proportion of metabolitally active organs (e.g., the liver makes up 6% of the THESTANDARDMETABOLICRATE
R226
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body mass of a mouse but only 1.8% of the body mass of a horse) and partially due to the metabolic activity of the organs (4, 7, 9). For instance, Krebs found that liver slices from a mouse consumed oxygen at 7.4 times the rate measured in liver slices from a horse. However, from their tissue slice studies, these workers (4, 7, 9) were unable to demonstrate whether the decrease in oxygen consumption rate with increasing body masswas due to 1) a decrease in the number of cells per unit mass of liver or 2) a decrease in the activity of the individual cells. In this study, we focus on oxygen consumption in hepatocytes freshly isolated from mammals with a 12,500-fold range in body mass, from 20-g mice to 250-kg horses. MATERIAL
AND
METHODS
Materiak. Stock 9% (wt/vol) bovine serum albumin was defatted by the method of Chen (3) and dialyzed against 153 mM NaCl and 11 mM KCl. All other materials are as described in Harper and Brand (5). IsoZation of hepatocytes. Hepatocytes were prepared essentially by the method of Seglen (12). Mouse and rat livers were perfused in situ. For larger mammals, lobes or partial lobes of liver were excised and cannulated. All mammals had been fed ad libitum. Mice and rats were killed under anesthetic; all other mammals were killed with a lethal dose of pentobarbitone. Incubation of hepatocytes. Cells were suspended in (in mM) 118 NaCl, 1.2 KH2P04, 1.2 MgS04, 4.75 KCl, 25 NaHC03, 5 N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid, and 2.5 CaClz and kept on ice before experimentation. Incubations of cells were carried out using 2.125 ml of incubation medium plus 0.375 ml of cell suspension (- 25 mg dry mass/ml) in ZO-ml stoppered glass vials at 37°C in a shaking water bath (100 cycles/min). The incubation medium contained (in mM) 106 NaCl, 5 KCl, 25 NaHC03, 0.41 MgS04, 10 NaBHP04, 2.5 CaC12, 10 glucose, 10 lactate, and 1 pyruvate as well as 2.25% (wt/vol> defatted b ovine serum albumin prior to addition of the cell suspension. The gas phase above the incubating cells was 95% air-5% CO2 to allow equilibration of the medium to a pH of 7.4. Cells were incubated at 37°C for 30 min before any measurements were made. Hepatocyte viability was routinely determined by exclusion of 0.6% (0.6 g/100 ml) trypan blue in 0.89% NaCl (0.89 g/100 ml). Only hepatocyte preparations with greater than or equal to 90% viability were used in this study. Hepatocyte volume measurements, measurement of resting hepatocyte oxygen consumption, cell number measurements, and electron microscopy are described in Harper and Brand (5). RESULTS
AND
DISCUSSION
Table 1 describes the nine species of mammals used in this study, the calculated standard metabolic rates, the
o 1995 the American
Physiological
Society
CELLULAR
Table 1. General properties from those mammals
of mammals
OXYGEN
CONSUMPTION
AND
BODY
used in the study and hepatocyte
R227
MASS
oxygen consumption
rates and volumes
Mammal Mouse
Gender Body mass, kg Calculated standard metabolic rate (293 - M”.7”) ’ kJ/day Calculated mass-specific metabolic rate kJ.day-l.kg’l (293 -M-0.25) Hepatocyte oxygen consumption rates, nmol 02.min+mg dry mass of cells- l Hepatocyte volume, ww dry mass of cells
Rat
F
F/M
(11) 0.27+0.02
0.02t0.00
Ferret
16
779
Rabbit
Sheep
M
F/M
(4) 1.2+0.1(10)
M
2.5*0.1(7)
30+4
Pig
F
(2)
33.8k0.4
Pig
F
Horse
F
(5) 37+3 (7)
F
160'" (3)
250’”
(3)
110
336
583
3,756
4,107
4,397
13,181
18,421
406
280
233
125
122
118
82
74
18 + 3 (4)
8.4kO.6 (4)
7.8d.O
1.9 + 0.5 (4)
1.2 + 0.2 (4)
1.5 + 0.2 (10)
Values are means +: SE; (n) = no. of mammals. F, female; least duplicate for each animal. *Approximate values.
(10) 2.3kO.3 (7) 5.221.7
1.3 t 0.2 (7)
M, male.
calculated mass-specific metabolic rates, the hepatocyte oxygen consumption rates, and the cell volumes for each of the mammalian groups. There was no significant correlation between hepatocyte volume per dry mass of cells (measured using radiolabeled water and inulin) and body mass. Nor was there a significant difference between the number of mouse hepatocytes [3.5 t 0.4 x lo5 (4 mice)] and the number of horse hepatocytes [3.5 t 0.1 x lo5 (3 h orses)] per milligram dry mass of cells. Taken together these observations would agree with the consensus of opinion in the literature that cell size doesnot vary with body mass(2,ll). However, a log-log plot of hepatoqte oxygen consumption as a function of body mass gave a significant inverse correlation with exponent - 0.18 (Fig. 1). In. general then we can say that with a 12,500-fold increase in body mass there is a 5.5-fold decrease in hepatocyte oxygen consumption rate. The exponent of -0.18 is similar to the value for liver slices obtained by Couture and Hulbert (4) of - 0.2 1 and practically identical to the exponent of - 0.17 that one gets when one plots log of oxygen consumption rate against log of body mass from the data in Table 7 of Krebs (9) (results not shown). We can therefore conclude that most if not all of the decrease in oxygen consumption rate in liver with increasing body mass is due to a marked decrease in metabolic activity of individual liver cells and a corresponding decrease in their activity per unit dry mass of cells and per unit mass of liver. In summary then, this study is the first to demonstrate directly that cells from larger mammals consume less oxygen than cells from smaller mammals. This novel finding explains and confirms the early observa-
(2)
2.2 + 0.1 (2)
Hepatocyte
oxygen
4.020.6
(5) 3.5kO.3 (7) 3.8d.5
1.4 + 0.3 (5) consumption
1.9 + 0.3 (7) rates
(3) 2.120.5
1.0 + 0.4 (3)
and volumes
were
(3)
1.5 + 0.4 (2)
measured
in at
tions made by Krebs (9) and later by Couture and Hulbert (4) using tissue slices. We can conclude that the decreased metabolic activity of the liver with increasing body mass is due to a decreased metabolic activity of the liver cells. Future work will determine the contribution to this effect made by differences in nonmitochondrial oxygen consumption, differences in mitochondrial number and size per cell, and differences in mitochondrial
g i5 .E "0
loo ..
: .
-0.18
y=6.83x r=-0.82:n = 9:p< 0.01
.
1 ’ ’ ’ m11’s” ’ ’ ’ ‘m’a” ’ ’ 1‘.Ol .1 1 10
’ ’ ’ L‘m’u 100
loo0
Body mass (kg) Fig. 1. Hepatocyte oxygen consumption as a function of Log-log plot of hepatocyte oxygen consumption rate as a body mass for data given in Table 1. The line through the fitted by linear regression. The slope of the line is -0.18 (P
body mass. function of points was < 0.01).
R228
CELLULAR
OXYGEN
CONSUMPTION
activity in situ, although the observed differences in proton leakiness of liver mitochondria isolated from different sized mammals (10) are likely to play a part. Address for reprint Univ. of Dublin, Trinity Received
26 October
requests: College,
R. K. Porter, Dept. of Biochemistry, Dublin 2, Republic of Ireland.
1994; accepted
in final
form
20 April
1995.
REFERENCES 1. Brody, S. Bioenergetics and Growth. New York: Reinhold, 1945. 2. Calder, W. A. Size, Function and Life History. Cambridge, MA: Harvard University Press, 1984. 3. Chen, R. F. Removal of fatty acids from serum albumin by charcoal treatment. J. Biol. Chem. 242: 173-181, 1967. 4. Couture, P., and A. J. Hulbert. On the relationship between body mass, tissue metabolic rate, and sodium pump activity in mammalian liver and kidney cortex. Am. J. PhysioZ. 268 (Regulatory Integrative Comp. PhysioZ. 37): R641-R650, 1995.
AND
BODY
MASS
5. Harper, M.-E., and M. D. Brand. The quantitative contributions 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. 6. Jansky, L. Adaptibility of heat production mechanisms in homeotherms.Acta Uniu. CaroZ. BioZ. 1: 1-91, 1965. 7. Kleiber, M. Body size and metabolism of liver slices in vitro. Proc. Sot. Exp. BioZ. Med. 48: 419-423, 1941. 8. Kleiber, M. The Fire of Life. New York: Wiley, 1961. 9. Krebs, H. A. Body mass and tissue respiration. Biochim. Biophys. Acta 4: 249-269, 1950. 10. Porter, R. K., and M. D. Brand. Body mass dependence of proton leak in mitochondria and its relevance to metabolic rate. Nature Lond. 362: 628-630,1993. 11. Schmidt-Nielsen, K. ScaZing: Why is AnimaZ Size so Important? Cambridge, UK: Cambridge University Press, 1984. 12. Seglen, P. 0. Preparation of isolated rat liver cells. Method CeZZ BioZ. 13: 29-83, 1976.
