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The nucleic acid content of skeletal muscle and liver in mammals of different body size
Comp. Biochem. Physiol., 1969, Vol. 28, pp. 897 to 905. Pergamon Press. Printed in Great Britain
T H E NUCLEIC ACID C O N T E N T OF SKELETAL MUSCLE AND LIVER IN MAMMALS OF D I F F E R E N T BODY SIZE* H. N. M U N R O and J. A. M. G R A Y Physiological Chemistry Laboratories, Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, and Department of Biochemistry, University of Glasgow, Glasgow, Scotland (Received 2 flune 1968)
A b s t r a c t - - 1 . The concentrations of RNA and of DNA have been measured
in the liver and posterior thigh muscles of a series of adult mammals varying in body weight from the mouse to the horse. 2. RNA concentration per 100 g tissue declines progressively in both tissues with increasing species size. 3. As body weight increases, DNA content decreases in muscle but not in liver, thus indicating that species size affect mean cell size of muscle but not of liver. 4. The total amounts of RNA and of DNA in the liver and in the whole skeletal musculature have been estimated. 5. The total RNA content of liver changes as the 0"77 power of body weight, in agreement with other parameters of protein metabolism, whereas the RNA content of muscle is related to the 0"89 power of body weight over the series of mammals. 6. In both tissues, total DNA content is related to the 0"91 power of body weight. INTRODUCTION MANe of the metabolic phenomena of mammals are known to be systematically affected by the body size of the species. T h u s it is well known that the basal energy metabolism of mammals is related to the 0.73 power of body weight, so that per kg of body weight, the basal metabolism of man is about one-fifth that of the rat (Brody, 1945). T h e intensity of protein metabolism per unit of body weight also diminishes progressively with increasing size of species. This is shown by a progressive reduction in larger mammals in (a) the amount of urinary N excreted per kg of body weight by animals eating a protein-free diet, (b) the rate of exchange of ~5N-labelled amino acids with tissue proteins, and (c) the turnover of plasma proteins (Munro, 1959). Since tissue nucleic acids are concerned with protein synthesis, it might be anticipated that this reduction in the intensity of protein metabolism and particularly of protein synthesis in larger mammals would be associated with lower concentrations of nucleic acids in their tissues. * This investigation was supported by Grant CA-08893 from the National Institutes of Health. 897
898
H. N. MUNRO AND J. A. M. GRAY
T h e literature already contains several estimates of tissue nucleic acid concentrations, but it is not possible to use these published results in order to make comparisons between different species because of the large discrepancies between different methods of estimating R N A and D N A (Hutchison & Munro, 1961). M u n r o & Downie (1964) accordingly applied a uniform procedure to measure the concentrations of nucleic acids in the livers of m a m m a l s over a wide range of body weights. W h e n the livers of m a m m a l s of increasing body size were analysed, a progressive reduction in the amount of R N A per unit of D N A f r o m 4.45 in mouse liver to 1.29 in the case of cow liver was observed. T h i s implies that there is less R N A per liver cell in the case of the larger species. I n addition, liver weight forms a smaller proportion of total b o d y weight in the larger mammals, so that, relative to body weight, the total amount of R N A in the liver diminishes even more extensively as one progresses up the scale of m a m m a l i a n size. A similar study was then m a d e of the R N A and D N A content of the thyroid gland of different m a m m a l s (Begg et al., 1965). Again, it was shown that the amount of R N A expressed per unit of tissue D N A or per unit of body weight diminishes with increasing size of species. Skeletal muscle represents the largest single tissue in the body of the m a m m a l . Unlike the liver and other viscera, it remains a constant proportion of body weight irrespective of the size of the animal (Munro, 1969). T h e present publication describes changes in the R N A and D N A content of skeletal muscle obtained from various m a m m a l s , and compares these data with nucleic acid estimations made on the livers of the same animals. MATERIALS AND M E T H O D S Animals and tissues The mammals selected for study varied from 30 g (mouse) to 690 kg (horse) and had been receiving a normal diet. All were young adult males. The smaller animals were killed in the laboratory and the tissues processed immediately, but the bullocks and horses were slaughtered elsewhere and the tissues were immediately excised and frozen in vessels surrounded by solid CO 2 and ethanol for transport to the laboratory. The tissues taken for analysis were samples of liver and of the mixed muscles from the posterior aspect of the thigh. In the case of mice and rats, tissue samples from several animals were pooled for analysis. In the case of other species, tissue samples from each animal were analysed separately.
Analysis of samples In the case of liver, the weighed sample was homogenized directly in a chilled Nelco Blendor with 19 vol. of ice-cold water. The skeletal muscle samples were weighed and then frozen hard in a beaker surrounded by solid CO~ and ethanol. The frozen blocks were cut finely with a scalpel and homogenized in an all-glass homogenizer of the Potter-Elvehjem type with 9 vol. of ice-cold water. This was found to give a more satisfactory uniform homogenate than mincing the fresh tissue with scissors and then homogenizing the product. The nucleic acid content of the homogenates was estimated by a modified SchmidtThannhauser procedure (Munro & Fleck, 1966). This involved extracting the tissue homogenate with cold 0"2 N perchloric acid to remove acid-soluble small molecules, followed by digestion of the remaining precipitate with 0"3 N KOH for 1 hr at 37°C to solubilize the tissue RNA with minimal release of peptides. The tissue protein and DNA were separated fromthe ribonucleotides by acidification of the digest and centrifuged down.
M U S C L E AND LIVER N U C L E I C ACIDS I N M A M M A L S
899
The amount of RNA in the tissue was obtained from the u.v. absorption of the supernatant at 260 m/~. The precipitate was dissolved in 0"1 N KOH and DNA was then estimated on the solution by the method of Ceriotti (1952). The main source of error in estimating RNA by the above method is the presence of u.v.absorbing peptides in the RNA fraction (Munro & Fleck, 1966). Examination of the u.v. spectrum of the RNA fraction obtained in this way from liver samples suggested no significant peptide contamination, but there was evidence of slight distortion of the spectrum by peptides in the case of muscle samples. The amount of peptide present in the RNA extract was therefore directly estimated by the method of Lowry et aL (1951), and its contribution to the absorption of the extract at 260 m/z was calculated on the basis that 1/~g protein/ml solution has an absorption of 0"0008 extinction units at 260 m/~ (Fleck & Munro, 1962). On ten liver samples, the peptide content of the RNA extract was found to average 10/zg/ml, equivalent to 2 per cent of the total u.v. absorption at 260 m/z. In a similar series of muscle samples, the peptide content of the RNA extract was found to be 29/zg/ml, which accounted for 9"6 per cent of the absorption at 260 m/z. Accordingly, the absorption at 260 m/z of the RNA extract from muscle samples was corrected for the amount of peptide found by the method of Lowry et al. (1951). The efficiency of the estimation of DNA was also evaluated. The spectrum of the colour obtained by applying the Ceriotti (1952) reaction to the DNA fractions from liver and muscle was compared with the spectrum obtained with a pure sample of calf thymus DNA, and found to be in satisfactory agreement. As a further check, the DNA fractions from liver and muscle samples were first extracted three times with 1 N perchloric acid for 20 rain at 70°C and the Ceriotti reaction was then applied to the combined extracts. The values obtained for DNA were similar to those found by applying the reaction directly to the alkaline solution of the DNA fraction, as described earlier. RESULTS Table 1 shows the concentrations of R N A and of D N A per g of fresh tissue in the livers and skeletal muscles of various mammals arranged in order of increasing body weight. T h e r e is a progressive reduction in R N A concentration in both tissues, so that the liver and muscles of the horse have one-third the level found in the corresponding tissues of the mouse. T h e concentration of R N A in liver is m u c h higher than in muscle, being about eight times greater throughout the range of species. This large difference between the two tissues is confirmed by other studies. Devi et al. (1963) used the less specific orcinol reaction to measure R N A concentrations in the liver and skeletal muscles of rats of different ages. T h e y found the values for very y o u n g rats to be high and to diminish rapidly with increasing age until the animals weighed more than 100 g, after which more constant levels were observed in both tissues. F r o m this point onwards, the concentration of R N A in liver was about eight times that in muscle, in agreement with Table 1. Unlike RNA, species size affected D N A concentration differently in the two tissues. T h e amount of D N A per 100 g of liver did not alter appreciably over the range of mammals. Since diploid nuclei in all mammals have a similar D N A content, namely 7 x 10 -12 g per nucleus (Vendrely, 1955), this finding means that the mass of the average liver cell per set of chromosomes must be fairly constant in mammals of different body size. This conclusion is not invalidated by the frequency of polyploidy in the liver cells. Epstein (1967) has shown that polyploid cells have proportionally more cytoplasm, so that the ratio of cell mass to D N A remains
Body weight (kg) 860 710 465 455 380 310
Liver 106 88 64 51 47 39
Muscle
RNA concentration (mg/100 g)
231 253 171 203 264 254
Liver
58 59 37 35 30 26
Muscle
D N A concentration (mg/100 g)
3"72 2"81 2"72 2"24 1"44 1"22
Liver
1'83 1-49 1"73 1"46 1.57 1-49
Muscle
R N A / D N A ratio
I N T H E LIVER AND POSTERIOR T H I G H MUSCLES OF VARIOUS M A M M A L S ARRANGED I N A S C E N D I N G ORDER OF BODY W E I G H T
0"03 0"16 1"5 25"5 450 690
DNA
36 9 7 2 3 2
AND
Mouse (Mus musculus) Rat (Rattus norvegicus) Rabbit ( Oryctolagus cuniculus) Dog (Canis familiaris) Bullock (Bos taurus) Horse (Equus caballus)
RNA
Number analysed
OF
Species
TABLE I--CONCENTRATIONS
.~
Z
0
;Z
O O
MUSCLE AND LIVER NUCLEIC ACIDS IN MAMMALS
901
unaffected by the presence of polypoidy in a liver cell population. Unlike the liver, D N A content per 100 g of muscle was less in larger mammals, so that the posterior thigh muscles of the horse had more than twice the cell mass per unit of D N A that was found in the corresponding muscles of the mouse. The ratio of RNA to D N A provides an index of the amount of RNA per diploid set of chromosomes. Table 1 shows that the R N A / D N A ratio of the liver underwent a progressive fall, in agreement with the earlier findings of Munro & Downie (1964), whereas the ratio in muscle did not alter appreciably over the range of species. This suggests that the RNA content of the liver cell is regulated in relation to the size of the animal, whereas the muscle cell apparently maintains the same amount of RNA irrespective of species. This regulation is presumably exercised at the level of RNA polymerase function in the nucleus. In addition to alterations in nucleic acid content, the relative sizes of different tissues of the body undergo progressive changes with increasing weight of the animal. Thus the liver accounts for more than 5 per cent of the body weight of the mature mouse, whereas the liver of a horse is only a little over 1 per cent of its body weight (Brody, 1945). On the other hand, a survey of published studies of the proportion of skeletal muscle in various adult mammals shows that the amount of muscle in the body is not systematically influenced by the final body size of the mammal (Munro, 1969). The regression equations of Brody (1945) for the proportion of liver and of Munro (1969) for skeletal muscle at different body sizes have therefore been used to estimate the percentage of liver and of muscle in the bodies of animals of the weights shown in Table 1. These estimates are reported in Table 2, which also gives the total RNA content of the liver and of the whole skeletal musculature per kg of the weight of the animal. These calculations are based on the assumption that the nucleic acid concentrations in the thigh muscles are representative of the skeletal musculature in general. Goldberg (1967) has compared red (tonic) muscles with white (plastic) muscles and has shown that the former are more active in protein synthesis and have a higher content of RNA. However, most muscles are composed of both red and white fibres and we have assumed that the posterior thigh muscles are a representative mixture whose composition undergoes changes reflecting those of the musculature as a whole. The data demonstrate that the total amount of RNA expressed per unit of body weight diminishes in both tissues as the size of species increases. However, the diminution is more rapid in the liver than in muscle. The total RNA content of mouse liver is similar to the amount of RNA estimated to be present in all its skeletal muscles, whereas the horse has more than four times the amount of RNA in its musculature than it has in its liver. On the other hand, calculation shows that the D N A content of liver and of muscle per kg of body weight diminishes more or less in parallel over the range of species shown in Table 2. This occurs because, although the D N A concentration in the liver remains constant per 100 g of tissue, liver mass forms a smaller proportion of body weight in larger mammals ; in contrast the total mass of muscle remains constant, but the concentration of DNA diminishes progressively with increasing species size.
Mouse Rat Rabbit Dog Bullock Horse
Species 53 42 30 21 14 13
Liver
450 450 450 450 450 450
Muscle
Relative weight of tissue (g/kg body wt.)
455 300 140 96 53 40
Liver
477 396 288 230 212 176
Muscle
1"0 1"3 2"0 2"4 4-0 4"4
Muscle/liver ratio
Total R N A in tissue (mg/kg body wt.)
WEIGHT
125 106 51 43 37 33
Liver
261 266 167 158 137 117
Muscle
2-1 2"5 3"3 3-7 3"7 3"6
Muscle/liver ratio
Total D N A in tissue (mg/kg body wt.)
TABLE 2--TOTAL AMOUNTS OF RNA AND DNA IN THE LIVER AND SKELETAL MUSCULATURE OF THE SAME SPECIES EXPRESSED PER kg BODY
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O
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b~
M U S C L E AND LIVER N U C L E I C ACIDS I N M A M M A L S
903
DISCUSSION From the data given in Table 1, it has been concluded that, irrespective of the body size of the mammal, the average liver cell has a cytoplasmic mass that is constant per set of diploid chromosomes, whereas the mean muscle cell increases twofold in cytoplasmic mass per unit of DNA with increasing body weight over the range of mammals studied. The constancy of mean liver cell size is consonant with direct measurements of liver cell dimensions in various species of mammal (Teissier, 1939). In the case of skeletal muscle, there is some conflict of evidence regarding the relationship between muscle fibre size and body size. Gauthier & Padykula (1966) measured the dimensions of the fibres of the diaphragm and observed a twofold increase in thickness from the smallest to the largest mammals. However, Joubert (1956) was unable to demonstrate any relationship between mature body size and the diameter of muscle fibres in the gastrocnemius muscles of rabbits, sheep, pigs and cattle. This does not necessarily mean that the mass of contractile tissue per muscle cell nucleus does not become greater in larger mammals. This could occur through a reduction in the relative number of nuclei in each muscle, as the data in Table 1 would suggest. It should also be remembered that some of the nuclei of muscle are associated with the connective tissue of the muscle. We do not know whether the proportion of connective tissue to contractile cells varies in the muscles of mammals of different sizes. The tendency for liver cells to remain of constant size in different species and for muscle cells to become larger in the large mammals corresponds to a similar phenomenon during growth of a single species, such as the rat (Goss, 1966; Miller, 1969; Munro, 1969). During growth, the viscera enlarge throughout the growing period mainly through an increase in cell number (hyperplasia), whereas the skeletal muscles finally enlarge through an increase in cell size (hypertrophy). From our data it would appear that, during the evolution of mammals, liver has also enlarged exclusively by hyperplasia whereas to some extent muscle has increased by hypertrophy. The total amounts of RNA and of DNA per kg body weight diminish in liver and in muscle as the size of the mammal increases (Table 2). Data of this type can also be calculated in the form of allometric equations, in which the log of the tissue constituent or metabolic measurement (M) is plotted against the log of body weight (W) to give the linear relationship log M = log a+b log W, where a and b are constants (Brody, 1945). The equation is usually written as M = a W b, in which the exponent b describes the effect of body weight on the tissue constituent or metabolic measurement. Table 3 shows data for the effect of species size on several such parameters of protein metabolism. These parameters change approximately as the 0.7 power of the weight of the animal. This means that the endogenous output of nitrogen, the total incorporation of 15N into body protein and the rate of turnover of two plasma proteins all become much less in proportion to body size as one progresses up the scale of species. This can be compared with the effect of species size on the total RNA content of liver and of skeletal muscle. The total RNA content of the liver per kg body weight falls in parallel with the other parameters of protein metabolism cited above. On the other hand, the total RNA
904
H . N . MUNROAND J. A. M. GRAY TABLE 3--THE
INFLUENCE OF M A M M A L I . ~ BODY SIZE ON PROTEIN METABOLISM
Measurement
Exponent b relating this measurement to body weight in M = a W b
Endogenous urinary N Total body protein synthesis Plasma albumin turnover Ceruloplasmin turnover Liver RNA Muscle RNA Liver DNA Muscle DNA
0"72 0"74 0'66 0"76 0"77 0-91 0"89 0"91
The first four entries are from Munro (1969). content of the skeletal musculature diminishes as the 0.91 power of body weight. This means that, as the size of the mammal increases, the amount of R N A in skeletal muscle falls less rapidly than in liver. Since R N A is closely associated with protein synthesis, this implies that the intensity of protein metabolism in muscle may be relatively more important than in the liver in larger mammals. T h e implications of this conclusion are discussed in greater detail elsewhere (Munro, 1969). REFERENCES BEOO D. J., McGlm~ E. M. & MUNROH. N. (1965) The protein and nucleic acid content of the thyroid glands of different mammals. Endocrinology 76, 171-177. BRODYS. (1945) Bioenergetics and Growth. Reinhold, New York. CERIOTTI G. (1952) A microchemical determination of desoxyribonucleic acid. J. Biol. Chem. 198, 297-303. DEVI A., MUKUNDhNM. A., SRIVASTAVAU. & SABK~mN. K. (1963) The effect of age on the variations of deoxyribonucleic acid, ribonucleic acid and total nucleotides in liver, brain and muscle of rat. ExplCell. Res. 32, 242-250. EPSTEIN C. J. (1967) Cell size, nuclear content and the development of polyploidy in the mammalian liver. Proe. Nat. Acad. Sci. U.S. 57, 327-334. FLECK A. & MUNRO H. N. (1962) The precision of ultraviolet absorption measurements in the Schmidt-Thannhauser procedure for nucleic acid estimation. Biochim. biophys. Acta 55, 571-583. GAUTHIER G. F. & PADYKULAH. A. (1966) Cytological studies of fiber types in skeletal muscle, ft. cell. Biol. 28, 333-354. GOLDBERC A. L. (1967) Protein synthesis in tonic and phasic skeletal muscles. Nature, Lond. 216, 1219-1220. Goss R. J. (1966) Hypertrophy versus hyperplasia. Science 153, 1615-1620. HUTCHISONW. C. & MUNROH. N. (1961) The quantitative measurement of nucleic acids in biological materials. Analyst 86, 768-813. JOUBERTD. M. (1956) An analysis of factors influencing post-natal growth and development of the muscle fibre, ft. Agric. Sci. 47, 59-102. LowRY O. H., ROSEBROUGHN. J., FARRA. L. & RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent, ft. Biol. Chem. 193, 265-275.
MUSCLE AND LIVER NUCLEIC ACIDS IN MAMMALS
905
MILLER S. A. (1969) Protein metabolism during growth and development. In Mammalian Protein Metabolism (Edited by MUNRO H. N.), Vol. III. Academic Press, New York. (In press.) MUNRO H. N. (1969) Evolution of protein metabolism in mammals. In Mammalian Protein Metabolism (Edited by MUNRO H. N.), Vol. III. Academic Press, New York. (In press.) MUNRO H. N. & DOWNIE E. D. (1964) Relationship of hver composition to intensity of protein metabolism in different mammals. Nature, Lond. 203, 603-604. MUNRO H. N. & FLECK A. (1966) The determination of nucleic acids. In Methods of Biochemical Analysis (Edited by GLmK D.), Vol. 14, pp. 113-176. Interscience and Wylie, New York. TEmSlER G. (1939) Biom6trie de la cellule animale et vgg6tale. Tabul. Biolog. 19, 1-36. VENDRELY R. (1955) The deoxyribonucleic acid content of the nucleus. In The Nucleic Acids (Edited by CHARaA~TE. & DAVIDSONJ. N.), Vol. II, pp. 155-180. Academic Press, New York.
T H E NUCLEIC ACID C O N T E N T OF SKELETAL MUSCLE AND LIVER IN MAMMALS OF D I F F E R E N T BODY SIZE* H. N. M U N R O and J. A. M. G R A Y Physiological Chemistry Laboratories, Department of Nutrition and Food Science, Massachusetts Institute of Technology, Cambridge, Massachusetts, and Department of Biochemistry, University of Glasgow, Glasgow, Scotland (Received 2 flune 1968)
A b s t r a c t - - 1 . The concentrations of RNA and of DNA have been measured
in the liver and posterior thigh muscles of a series of adult mammals varying in body weight from the mouse to the horse. 2. RNA concentration per 100 g tissue declines progressively in both tissues with increasing species size. 3. As body weight increases, DNA content decreases in muscle but not in liver, thus indicating that species size affect mean cell size of muscle but not of liver. 4. The total amounts of RNA and of DNA in the liver and in the whole skeletal musculature have been estimated. 5. The total RNA content of liver changes as the 0"77 power of body weight, in agreement with other parameters of protein metabolism, whereas the RNA content of muscle is related to the 0"89 power of body weight over the series of mammals. 6. In both tissues, total DNA content is related to the 0"91 power of body weight. INTRODUCTION MANe of the metabolic phenomena of mammals are known to be systematically affected by the body size of the species. T h u s it is well known that the basal energy metabolism of mammals is related to the 0.73 power of body weight, so that per kg of body weight, the basal metabolism of man is about one-fifth that of the rat (Brody, 1945). T h e intensity of protein metabolism per unit of body weight also diminishes progressively with increasing size of species. This is shown by a progressive reduction in larger mammals in (a) the amount of urinary N excreted per kg of body weight by animals eating a protein-free diet, (b) the rate of exchange of ~5N-labelled amino acids with tissue proteins, and (c) the turnover of plasma proteins (Munro, 1959). Since tissue nucleic acids are concerned with protein synthesis, it might be anticipated that this reduction in the intensity of protein metabolism and particularly of protein synthesis in larger mammals would be associated with lower concentrations of nucleic acids in their tissues. * This investigation was supported by Grant CA-08893 from the National Institutes of Health. 897
898
H. N. MUNRO AND J. A. M. GRAY
T h e literature already contains several estimates of tissue nucleic acid concentrations, but it is not possible to use these published results in order to make comparisons between different species because of the large discrepancies between different methods of estimating R N A and D N A (Hutchison & Munro, 1961). M u n r o & Downie (1964) accordingly applied a uniform procedure to measure the concentrations of nucleic acids in the livers of m a m m a l s over a wide range of body weights. W h e n the livers of m a m m a l s of increasing body size were analysed, a progressive reduction in the amount of R N A per unit of D N A f r o m 4.45 in mouse liver to 1.29 in the case of cow liver was observed. T h i s implies that there is less R N A per liver cell in the case of the larger species. I n addition, liver weight forms a smaller proportion of total b o d y weight in the larger mammals, so that, relative to body weight, the total amount of R N A in the liver diminishes even more extensively as one progresses up the scale of m a m m a l i a n size. A similar study was then m a d e of the R N A and D N A content of the thyroid gland of different m a m m a l s (Begg et al., 1965). Again, it was shown that the amount of R N A expressed per unit of tissue D N A or per unit of body weight diminishes with increasing size of species. Skeletal muscle represents the largest single tissue in the body of the m a m m a l . Unlike the liver and other viscera, it remains a constant proportion of body weight irrespective of the size of the animal (Munro, 1969). T h e present publication describes changes in the R N A and D N A content of skeletal muscle obtained from various m a m m a l s , and compares these data with nucleic acid estimations made on the livers of the same animals. MATERIALS AND M E T H O D S Animals and tissues The mammals selected for study varied from 30 g (mouse) to 690 kg (horse) and had been receiving a normal diet. All were young adult males. The smaller animals were killed in the laboratory and the tissues processed immediately, but the bullocks and horses were slaughtered elsewhere and the tissues were immediately excised and frozen in vessels surrounded by solid CO 2 and ethanol for transport to the laboratory. The tissues taken for analysis were samples of liver and of the mixed muscles from the posterior aspect of the thigh. In the case of mice and rats, tissue samples from several animals were pooled for analysis. In the case of other species, tissue samples from each animal were analysed separately.
Analysis of samples In the case of liver, the weighed sample was homogenized directly in a chilled Nelco Blendor with 19 vol. of ice-cold water. The skeletal muscle samples were weighed and then frozen hard in a beaker surrounded by solid CO~ and ethanol. The frozen blocks were cut finely with a scalpel and homogenized in an all-glass homogenizer of the Potter-Elvehjem type with 9 vol. of ice-cold water. This was found to give a more satisfactory uniform homogenate than mincing the fresh tissue with scissors and then homogenizing the product. The nucleic acid content of the homogenates was estimated by a modified SchmidtThannhauser procedure (Munro & Fleck, 1966). This involved extracting the tissue homogenate with cold 0"2 N perchloric acid to remove acid-soluble small molecules, followed by digestion of the remaining precipitate with 0"3 N KOH for 1 hr at 37°C to solubilize the tissue RNA with minimal release of peptides. The tissue protein and DNA were separated fromthe ribonucleotides by acidification of the digest and centrifuged down.
M U S C L E AND LIVER N U C L E I C ACIDS I N M A M M A L S
899
The amount of RNA in the tissue was obtained from the u.v. absorption of the supernatant at 260 m/~. The precipitate was dissolved in 0"1 N KOH and DNA was then estimated on the solution by the method of Ceriotti (1952). The main source of error in estimating RNA by the above method is the presence of u.v.absorbing peptides in the RNA fraction (Munro & Fleck, 1966). Examination of the u.v. spectrum of the RNA fraction obtained in this way from liver samples suggested no significant peptide contamination, but there was evidence of slight distortion of the spectrum by peptides in the case of muscle samples. The amount of peptide present in the RNA extract was therefore directly estimated by the method of Lowry et aL (1951), and its contribution to the absorption of the extract at 260 m/z was calculated on the basis that 1/~g protein/ml solution has an absorption of 0"0008 extinction units at 260 m/~ (Fleck & Munro, 1962). On ten liver samples, the peptide content of the RNA extract was found to average 10/zg/ml, equivalent to 2 per cent of the total u.v. absorption at 260 m/z. In a similar series of muscle samples, the peptide content of the RNA extract was found to be 29/zg/ml, which accounted for 9"6 per cent of the absorption at 260 m/z. Accordingly, the absorption at 260 m/z of the RNA extract from muscle samples was corrected for the amount of peptide found by the method of Lowry et al. (1951). The efficiency of the estimation of DNA was also evaluated. The spectrum of the colour obtained by applying the Ceriotti (1952) reaction to the DNA fractions from liver and muscle was compared with the spectrum obtained with a pure sample of calf thymus DNA, and found to be in satisfactory agreement. As a further check, the DNA fractions from liver and muscle samples were first extracted three times with 1 N perchloric acid for 20 rain at 70°C and the Ceriotti reaction was then applied to the combined extracts. The values obtained for DNA were similar to those found by applying the reaction directly to the alkaline solution of the DNA fraction, as described earlier. RESULTS Table 1 shows the concentrations of R N A and of D N A per g of fresh tissue in the livers and skeletal muscles of various mammals arranged in order of increasing body weight. T h e r e is a progressive reduction in R N A concentration in both tissues, so that the liver and muscles of the horse have one-third the level found in the corresponding tissues of the mouse. T h e concentration of R N A in liver is m u c h higher than in muscle, being about eight times greater throughout the range of species. This large difference between the two tissues is confirmed by other studies. Devi et al. (1963) used the less specific orcinol reaction to measure R N A concentrations in the liver and skeletal muscles of rats of different ages. T h e y found the values for very y o u n g rats to be high and to diminish rapidly with increasing age until the animals weighed more than 100 g, after which more constant levels were observed in both tissues. F r o m this point onwards, the concentration of R N A in liver was about eight times that in muscle, in agreement with Table 1. Unlike RNA, species size affected D N A concentration differently in the two tissues. T h e amount of D N A per 100 g of liver did not alter appreciably over the range of mammals. Since diploid nuclei in all mammals have a similar D N A content, namely 7 x 10 -12 g per nucleus (Vendrely, 1955), this finding means that the mass of the average liver cell per set of chromosomes must be fairly constant in mammals of different body size. This conclusion is not invalidated by the frequency of polyploidy in the liver cells. Epstein (1967) has shown that polyploid cells have proportionally more cytoplasm, so that the ratio of cell mass to D N A remains
Body weight (kg) 860 710 465 455 380 310
Liver 106 88 64 51 47 39
Muscle
RNA concentration (mg/100 g)
231 253 171 203 264 254
Liver
58 59 37 35 30 26
Muscle
D N A concentration (mg/100 g)
3"72 2"81 2"72 2"24 1"44 1"22
Liver
1'83 1-49 1"73 1"46 1.57 1-49
Muscle
R N A / D N A ratio
I N T H E LIVER AND POSTERIOR T H I G H MUSCLES OF VARIOUS M A M M A L S ARRANGED I N A S C E N D I N G ORDER OF BODY W E I G H T
0"03 0"16 1"5 25"5 450 690
DNA
36 9 7 2 3 2
AND
Mouse (Mus musculus) Rat (Rattus norvegicus) Rabbit ( Oryctolagus cuniculus) Dog (Canis familiaris) Bullock (Bos taurus) Horse (Equus caballus)
RNA
Number analysed
OF
Species
TABLE I--CONCENTRATIONS
.~
Z
0
;Z
O O
MUSCLE AND LIVER NUCLEIC ACIDS IN MAMMALS
901
unaffected by the presence of polypoidy in a liver cell population. Unlike the liver, D N A content per 100 g of muscle was less in larger mammals, so that the posterior thigh muscles of the horse had more than twice the cell mass per unit of D N A that was found in the corresponding muscles of the mouse. The ratio of RNA to D N A provides an index of the amount of RNA per diploid set of chromosomes. Table 1 shows that the R N A / D N A ratio of the liver underwent a progressive fall, in agreement with the earlier findings of Munro & Downie (1964), whereas the ratio in muscle did not alter appreciably over the range of species. This suggests that the RNA content of the liver cell is regulated in relation to the size of the animal, whereas the muscle cell apparently maintains the same amount of RNA irrespective of species. This regulation is presumably exercised at the level of RNA polymerase function in the nucleus. In addition to alterations in nucleic acid content, the relative sizes of different tissues of the body undergo progressive changes with increasing weight of the animal. Thus the liver accounts for more than 5 per cent of the body weight of the mature mouse, whereas the liver of a horse is only a little over 1 per cent of its body weight (Brody, 1945). On the other hand, a survey of published studies of the proportion of skeletal muscle in various adult mammals shows that the amount of muscle in the body is not systematically influenced by the final body size of the mammal (Munro, 1969). The regression equations of Brody (1945) for the proportion of liver and of Munro (1969) for skeletal muscle at different body sizes have therefore been used to estimate the percentage of liver and of muscle in the bodies of animals of the weights shown in Table 1. These estimates are reported in Table 2, which also gives the total RNA content of the liver and of the whole skeletal musculature per kg of the weight of the animal. These calculations are based on the assumption that the nucleic acid concentrations in the thigh muscles are representative of the skeletal musculature in general. Goldberg (1967) has compared red (tonic) muscles with white (plastic) muscles and has shown that the former are more active in protein synthesis and have a higher content of RNA. However, most muscles are composed of both red and white fibres and we have assumed that the posterior thigh muscles are a representative mixture whose composition undergoes changes reflecting those of the musculature as a whole. The data demonstrate that the total amount of RNA expressed per unit of body weight diminishes in both tissues as the size of species increases. However, the diminution is more rapid in the liver than in muscle. The total RNA content of mouse liver is similar to the amount of RNA estimated to be present in all its skeletal muscles, whereas the horse has more than four times the amount of RNA in its musculature than it has in its liver. On the other hand, calculation shows that the D N A content of liver and of muscle per kg of body weight diminishes more or less in parallel over the range of species shown in Table 2. This occurs because, although the D N A concentration in the liver remains constant per 100 g of tissue, liver mass forms a smaller proportion of body weight in larger mammals ; in contrast the total mass of muscle remains constant, but the concentration of DNA diminishes progressively with increasing species size.
Mouse Rat Rabbit Dog Bullock Horse
Species 53 42 30 21 14 13
Liver
450 450 450 450 450 450
Muscle
Relative weight of tissue (g/kg body wt.)
455 300 140 96 53 40
Liver
477 396 288 230 212 176
Muscle
1"0 1"3 2"0 2"4 4-0 4"4
Muscle/liver ratio
Total R N A in tissue (mg/kg body wt.)
WEIGHT
125 106 51 43 37 33
Liver
261 266 167 158 137 117
Muscle
2-1 2"5 3"3 3-7 3"7 3"6
Muscle/liver ratio
Total D N A in tissue (mg/kg body wt.)
TABLE 2--TOTAL AMOUNTS OF RNA AND DNA IN THE LIVER AND SKELETAL MUSCULATURE OF THE SAME SPECIES EXPRESSED PER kg BODY
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M U S C L E AND LIVER N U C L E I C ACIDS I N M A M M A L S
903
DISCUSSION From the data given in Table 1, it has been concluded that, irrespective of the body size of the mammal, the average liver cell has a cytoplasmic mass that is constant per set of diploid chromosomes, whereas the mean muscle cell increases twofold in cytoplasmic mass per unit of DNA with increasing body weight over the range of mammals studied. The constancy of mean liver cell size is consonant with direct measurements of liver cell dimensions in various species of mammal (Teissier, 1939). In the case of skeletal muscle, there is some conflict of evidence regarding the relationship between muscle fibre size and body size. Gauthier & Padykula (1966) measured the dimensions of the fibres of the diaphragm and observed a twofold increase in thickness from the smallest to the largest mammals. However, Joubert (1956) was unable to demonstrate any relationship between mature body size and the diameter of muscle fibres in the gastrocnemius muscles of rabbits, sheep, pigs and cattle. This does not necessarily mean that the mass of contractile tissue per muscle cell nucleus does not become greater in larger mammals. This could occur through a reduction in the relative number of nuclei in each muscle, as the data in Table 1 would suggest. It should also be remembered that some of the nuclei of muscle are associated with the connective tissue of the muscle. We do not know whether the proportion of connective tissue to contractile cells varies in the muscles of mammals of different sizes. The tendency for liver cells to remain of constant size in different species and for muscle cells to become larger in the large mammals corresponds to a similar phenomenon during growth of a single species, such as the rat (Goss, 1966; Miller, 1969; Munro, 1969). During growth, the viscera enlarge throughout the growing period mainly through an increase in cell number (hyperplasia), whereas the skeletal muscles finally enlarge through an increase in cell size (hypertrophy). From our data it would appear that, during the evolution of mammals, liver has also enlarged exclusively by hyperplasia whereas to some extent muscle has increased by hypertrophy. The total amounts of RNA and of DNA per kg body weight diminish in liver and in muscle as the size of the mammal increases (Table 2). Data of this type can also be calculated in the form of allometric equations, in which the log of the tissue constituent or metabolic measurement (M) is plotted against the log of body weight (W) to give the linear relationship log M = log a+b log W, where a and b are constants (Brody, 1945). The equation is usually written as M = a W b, in which the exponent b describes the effect of body weight on the tissue constituent or metabolic measurement. Table 3 shows data for the effect of species size on several such parameters of protein metabolism. These parameters change approximately as the 0.7 power of the weight of the animal. This means that the endogenous output of nitrogen, the total incorporation of 15N into body protein and the rate of turnover of two plasma proteins all become much less in proportion to body size as one progresses up the scale of species. This can be compared with the effect of species size on the total RNA content of liver and of skeletal muscle. The total RNA content of the liver per kg body weight falls in parallel with the other parameters of protein metabolism cited above. On the other hand, the total RNA
904
H . N . MUNROAND J. A. M. GRAY TABLE 3--THE
INFLUENCE OF M A M M A L I . ~ BODY SIZE ON PROTEIN METABOLISM
Measurement
Exponent b relating this measurement to body weight in M = a W b
Endogenous urinary N Total body protein synthesis Plasma albumin turnover Ceruloplasmin turnover Liver RNA Muscle RNA Liver DNA Muscle DNA
0"72 0"74 0'66 0"76 0"77 0-91 0"89 0"91
The first four entries are from Munro (1969). content of the skeletal musculature diminishes as the 0.91 power of body weight. This means that, as the size of the mammal increases, the amount of R N A in skeletal muscle falls less rapidly than in liver. Since R N A is closely associated with protein synthesis, this implies that the intensity of protein metabolism in muscle may be relatively more important than in the liver in larger mammals. T h e implications of this conclusion are discussed in greater detail elsewhere (Munro, 1969). REFERENCES BEOO D. J., McGlm~ E. M. & MUNROH. N. (1965) The protein and nucleic acid content of the thyroid glands of different mammals. Endocrinology 76, 171-177. BRODYS. (1945) Bioenergetics and Growth. Reinhold, New York. CERIOTTI G. (1952) A microchemical determination of desoxyribonucleic acid. J. Biol. Chem. 198, 297-303. DEVI A., MUKUNDhNM. A., SRIVASTAVAU. & SABK~mN. K. (1963) The effect of age on the variations of deoxyribonucleic acid, ribonucleic acid and total nucleotides in liver, brain and muscle of rat. ExplCell. Res. 32, 242-250. EPSTEIN C. J. (1967) Cell size, nuclear content and the development of polyploidy in the mammalian liver. Proe. Nat. Acad. Sci. U.S. 57, 327-334. FLECK A. & MUNRO H. N. (1962) The precision of ultraviolet absorption measurements in the Schmidt-Thannhauser procedure for nucleic acid estimation. Biochim. biophys. Acta 55, 571-583. GAUTHIER G. F. & PADYKULAH. A. (1966) Cytological studies of fiber types in skeletal muscle, ft. cell. Biol. 28, 333-354. GOLDBERC A. L. (1967) Protein synthesis in tonic and phasic skeletal muscles. Nature, Lond. 216, 1219-1220. Goss R. J. (1966) Hypertrophy versus hyperplasia. Science 153, 1615-1620. HUTCHISONW. C. & MUNROH. N. (1961) The quantitative measurement of nucleic acids in biological materials. Analyst 86, 768-813. JOUBERTD. M. (1956) An analysis of factors influencing post-natal growth and development of the muscle fibre, ft. Agric. Sci. 47, 59-102. LowRY O. H., ROSEBROUGHN. J., FARRA. L. & RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent, ft. Biol. Chem. 193, 265-275.
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MILLER S. A. (1969) Protein metabolism during growth and development. In Mammalian Protein Metabolism (Edited by MUNRO H. N.), Vol. III. Academic Press, New York. (In press.) MUNRO H. N. (1969) Evolution of protein metabolism in mammals. In Mammalian Protein Metabolism (Edited by MUNRO H. N.), Vol. III. Academic Press, New York. (In press.) MUNRO H. N. & DOWNIE E. D. (1964) Relationship of hver composition to intensity of protein metabolism in different mammals. Nature, Lond. 203, 603-604. MUNRO H. N. & FLECK A. (1966) The determination of nucleic acids. In Methods of Biochemical Analysis (Edited by GLmK D.), Vol. 14, pp. 113-176. Interscience and Wylie, New York. TEmSlER G. (1939) Biom6trie de la cellule animale et vgg6tale. Tabul. Biolog. 19, 1-36. VENDRELY R. (1955) The deoxyribonucleic acid content of the nucleus. In The Nucleic Acids (Edited by CHARaA~TE. & DAVIDSONJ. N.), Vol. II, pp. 155-180. Academic Press, New York.