rats was produced by feeding a proteindeficient diet to the mothers, it i s probably inappropriate to attribute this to protein deficiency in the young animals. The limitation of protein in lactating animals probably does not produce a proteindeficient milk but rather produces less milk. Thus the result in the young animals is similar to that produced by large litters compared to small litters rather than prot e i n deficiency per se in the young animals. 0
1. Protein Deficiency and Tooth and Salivary Gland Development. Nutrition Reviews 32: 24-27, 1974 2. L. Menaker and J. M. Navia: Effect of Undernutrition During the Perinatal Period on Caries Development in the Rat. V. Changes in Whole Saliva Volume and Protein Content. J. Dent. Res. 53: 592-597,1974 3. M. C. Madeira, S. Hetem, and M. A. Rulli: Relationship Between the Number of R a t Littermates per Dam and Mandibular Incisor Growth. J. Dent. Res. 53: 634636. 1974
ADAPTATION TO LOW PROTEIN INTAKES In rats urea nitrogen changes rapidly in response to the protein intake. A most striking phenomenon is that key hepatic enzymes involved in nitrogen disposition change in concert and to similar degrees in response to the alterations in diet. Key Words: urea, urinary nitrogen, enzymes, casein, protein intake, half-lives
It is well accepted that nitrogen output falls as intake is reduced, but the mechanisms underlying this simple but fundamental process are poorly understood. It is known that the major component of urinary nitrogen is usually urea. It has also been established that in malnourished children urea as a fraction of urinary nitrogen i s depressed.2 The rate a t which these changes take place i s not clear and an investigation of the mechanisms involved cannot be obtained with human subjects. It has been shown that when the protein intake is markedly reduced the urinary excretion stabilizes in adult men after approximately six to ten days3 and in the infant in approximately two days4 Das and Waterlow5 used young rats, altered their nitrogen intake, and studied the nitrogen output and the changes in hepatic enzymes which are concerned with urea metabolism directly or indirectly. The enzymes they studied were the urea cycle enzymes arginase, argi ninosuccinate Iyase, and argininosuccinate synthetase as well as glutamate dehydrogenase and two transaminases, alanine aminotransferase, and aspartate aminotransferase. The last three were chosen because they make amino groups available for entry into the urea cycle. 180 NUTRITION REVIEWSNOL. 33, NO. 6 / J U N E 1975
In the first major experiment, the effect of reducing the casein content of the diet was studied. Rats were changed from a high (135 g of casein per kilogram) to a low (45 g of casein per kilogram) protein diet and urine collected in six hourly periods. It was found that urinary nitrogen reached the level appropriate for the low intake after 24 to 30 hours. Rats were sacrificed a t appropriate intervals and the six hepatic enzymes which originally showed a high activity on the high protein diet, then decreased and reached their new level in about the same time. The striking phenomenon was that all six enzymes changed a t the same rates and to a similar degree. When the lowest activities of the six enzymes were expressed as a percent of the value on the high protein diet, a t 30 hours they ranged from 32 to 39.5 percent. In control rats maintained on the low protein diet, the same enzymes were between 30.5 and 37 percent of the control value. It is thus clear that the enzyme levels had adapted to the level appropriate for the specific diet. The converse experiment was then done. Rats were maintained on the low protein diet for one week, then some were changed to the high protein diet. Again, the urinary nitrogen reached the appropriate plateau in
about 30 hours. Rats were sacrificed in groups a t regular intervals after the change to the high protein diet. By 32 hours all six enzymes had almost reached the levels appropriate for that protein intake. The data from these two experiments were combined, and by plotting the rates of change of the total enzyme activity with time, it was possible t o calculate the halflives of the various enzymes. The half-lives were all remarkably similar, and were the same if the calculations were based on the experiments in which the enzyme activities were increasing or decreasing. Since the half-lives were the same, irrespective of whether the enzyme activity was increasing or decreasing, the authors contend that the changes were caused by a change in the rate of synthesis. To investigate the possibility that it was simply the nitrogen output which affected the enzyme levels, rats were fed gelatin instead of casein t o produce an increase in urinary nitrogen. In these experiments, although the urinary nitrogen did increase, the enzyme activities in the liver actually fell slightly. Body weight also appeared t o fall. In the converse experiment in which rats were changed from the diet with gelatin to the low protein diet, urinary nitrogen fell and the activity of the liver enzymes did not change. When nitrogen intake was related t o output in rats on the casein diet, there was an excellent linear correlation, with a slope indicating that a quantity of nitrogen was excreted in the urine equivalent t o half of the amount ingested. With gelatin there were fewer experiments, but there was still a linear correlation between urinary nitrogen and nitrogen intake. Therefore, this study shows that young rats can rapidly reduce their urinary nitrogen in response t o lowering dietary nitrogen, and within 30 hours a plateau i s reached. This provides new information, but it i s not as significant as the demonstration that a wide range of enzymes change in concert and a t about the same rate in response t o dietary manipulations. It i s striking that the enzymes measured differ in their intracellular location, and certainly
in the case of the urea cycle enzymes, differ also in basal activity. As the authors point out, the change in enzyme activity cannot be a reflection simply of an increase in urea output since increasing urea output by feeding gelatin did not cause a rise in the enzyme activity. The fact that urea excretion did change without a change in urea cycle enzymes is taken t o indicate that the "potential capacity" of the enzymes i s not fully utilized. Caution should be expressed here. The conditions under which the enzyme i s assayed in vitro obviously may have little relationship t o i t s activity in vivo since levels of such factors as substrate, co-factors, and pH will be different. I n those experiments in which the enzyme activities increase, could the effect be a nonspecific one of increased protein intake enhancing protein synthesis generally? In that case several other enzymes should show increases in activity. However, a nonspecific rise in soluble protein appears unlikely. With a change in dietary casein from 40 t o 135 g per kilogram, the total hepatic arginase activity rose some fourfold and aspartate aminotransferase rose similarly. It is unlikely that soluble liver protein wou Id increase four-fold. The authors considered the possibility that hormones might be the signal for the change in enzyme activity and referred t o the work of McLean and Gurney6 which showed that cortisol increased urea cycle enzymes. Here one should point out that in adrenalectomized rats those authors found that although the urea cycle enzymes changed, their degree of change was variable, and the aspartate aminotransferase activity did not change. S ~ h i m k e , how~ ever, has proven that steriods are not the signal for the changes in urea cycle enzymes in response to dietary alterations. He showed convincingly that the enzyme proteins in the two dietary states are identical and have identical kinetic characteristics. He also showed that the change in activity i s not simply due to presence of activators or absence of inhibitors. There is simply more enzyme protein. A fundamental difference between Schimke's work and the NUTRITION REVIEWWVOL. 33.
NO. 6 / JUNE
1975
181
present one is that the enzyme half-lives are so widely different. Schimke showed a half-life for arginase of about five days,8 as compared with eight hours in the present study. There obviously cannot be such a difference in enzyme protein in the two strains of rats. The reason for these differences is not clear, but perhaps relates to the different methods used, especially for calculations of enzyme half-lives. It must be confessed that there is no clue from this or other studies as to why these enzymes should change in concert. It is hard to believe that there is a single genome for all six enzymes. The pathway for which the single genome theory was proposed with most vigor i s gluc~neogenesis.~We know now that although some enzymes with widely differing specific activities do change together in response to some stimuli, there are some in the pathway which do not change, and the degree of change of those that do can be widely varied. It is hoped that the authors will pursue these studies, perhaps measuring the levels of the key intermediates t o determine if and how they do change, and measuring other enzymes as well. (Their data on glutamate dehydrogenase are in conflict with other studies.) The problem of the dietary control of enzyme synthesis and degradation is fundamental in a general physiological sense. We should know what signals the shunting of amino acids into the synthetic pathway and not into the pathway for degradation and/or excretion as a product which i s normally not reutilizable to any great extent. In situations when protein is present in short supply the form of control could be important for therapeutic reasons. There may be conditions such as
182
NUTRITION REVIEWSIVOL. 33, NO. 6 I JUNE 1975
after surgery or during hyperalimentation in which it may be desirable to conserve nitrogen by reducing the amount entering the pathway for degradation. It is not too naive to suggest that if we knew the mechanism of control it might be possible to adjust it. 0 1. J. C. Waterlow: The Partition of Nitrogen in the Urine of Malnourished Jamaican Infants. Am. J. Clin. Nutrition 12: 235-240, 1963 2. J. M. L. Stephen and J. C. Waterlow: Effect of Malnutrition on Activity of Two Enzymes Concerned with Amino Acid Metabolism in Human Liver. Lancet I: 118-119, 1968 3. N. S. Scrimshaw, M. A. Hussein, E. Murray, W. M. Raud, and V. R. Young: Protein Requirements of Man: Variations in Obligatory Urinary and Fecal Nitrogen Losses in Young Men. J. Nutrition 102: 1595-1604, 1972 4. H. Chan: Adaptation of Urinary Nitrogen Excretion in Infants to Changes in Protein Intake. Brit. J. Nutrition 22: 315-323, 1968 5. T. K. Das and J. C. Waterlow: The Rate of A d a p t a t i o n o f Urea Cycle Enzymes, Aminotransferases and Glutamate Dehydrogenase to Changes in Dietary Protein Intake. Brit. J. Nutrition 32: 353-373, 1974 6. P. McLean and M. W. Gurney: Effect of Adrenalectomy and of Growth Hormones on Enzymes Concerned with Urea Synthesis in R a t Liver. Biochem J. 87: 96-104, 1963 7. R. T. Schimke: Adaptive Characteristics of Urea Cycle Enzymes in the Rat. J. Biol. Chem. 237: 459-468, 1962 8. R . T. Schimke: The importance of Both Synthesis and Degradation in the Control of Arginase Levels in R a t Liver. J. Biol. Chem. 239: 3808-3817, 1964 9. G. Weber, R. L. Singhal, N. B. Stamm, E. A. Fisher, and M. A. Mentendiek: Regulation of Enzymes Involved in Gluconeogenesis. Adv. Enzyme Reg. 2: 1-38, 1964
1. Protein Deficiency and Tooth and Salivary Gland Development. Nutrition Reviews 32: 24-27, 1974 2. L. Menaker and J. M. Navia: Effect of Undernutrition During the Perinatal Period on Caries Development in the Rat. V. Changes in Whole Saliva Volume and Protein Content. J. Dent. Res. 53: 592-597,1974 3. M. C. Madeira, S. Hetem, and M. A. Rulli: Relationship Between the Number of R a t Littermates per Dam and Mandibular Incisor Growth. J. Dent. Res. 53: 634636. 1974
ADAPTATION TO LOW PROTEIN INTAKES In rats urea nitrogen changes rapidly in response to the protein intake. A most striking phenomenon is that key hepatic enzymes involved in nitrogen disposition change in concert and to similar degrees in response to the alterations in diet. Key Words: urea, urinary nitrogen, enzymes, casein, protein intake, half-lives
It is well accepted that nitrogen output falls as intake is reduced, but the mechanisms underlying this simple but fundamental process are poorly understood. It is known that the major component of urinary nitrogen is usually urea. It has also been established that in malnourished children urea as a fraction of urinary nitrogen i s depressed.2 The rate a t which these changes take place i s not clear and an investigation of the mechanisms involved cannot be obtained with human subjects. It has been shown that when the protein intake is markedly reduced the urinary excretion stabilizes in adult men after approximately six to ten days3 and in the infant in approximately two days4 Das and Waterlow5 used young rats, altered their nitrogen intake, and studied the nitrogen output and the changes in hepatic enzymes which are concerned with urea metabolism directly or indirectly. The enzymes they studied were the urea cycle enzymes arginase, argi ninosuccinate Iyase, and argininosuccinate synthetase as well as glutamate dehydrogenase and two transaminases, alanine aminotransferase, and aspartate aminotransferase. The last three were chosen because they make amino groups available for entry into the urea cycle. 180 NUTRITION REVIEWSNOL. 33, NO. 6 / J U N E 1975
In the first major experiment, the effect of reducing the casein content of the diet was studied. Rats were changed from a high (135 g of casein per kilogram) to a low (45 g of casein per kilogram) protein diet and urine collected in six hourly periods. It was found that urinary nitrogen reached the level appropriate for the low intake after 24 to 30 hours. Rats were sacrificed a t appropriate intervals and the six hepatic enzymes which originally showed a high activity on the high protein diet, then decreased and reached their new level in about the same time. The striking phenomenon was that all six enzymes changed a t the same rates and to a similar degree. When the lowest activities of the six enzymes were expressed as a percent of the value on the high protein diet, a t 30 hours they ranged from 32 to 39.5 percent. In control rats maintained on the low protein diet, the same enzymes were between 30.5 and 37 percent of the control value. It is thus clear that the enzyme levels had adapted to the level appropriate for the specific diet. The converse experiment was then done. Rats were maintained on the low protein diet for one week, then some were changed to the high protein diet. Again, the urinary nitrogen reached the appropriate plateau in
about 30 hours. Rats were sacrificed in groups a t regular intervals after the change to the high protein diet. By 32 hours all six enzymes had almost reached the levels appropriate for that protein intake. The data from these two experiments were combined, and by plotting the rates of change of the total enzyme activity with time, it was possible t o calculate the halflives of the various enzymes. The half-lives were all remarkably similar, and were the same if the calculations were based on the experiments in which the enzyme activities were increasing or decreasing. Since the half-lives were the same, irrespective of whether the enzyme activity was increasing or decreasing, the authors contend that the changes were caused by a change in the rate of synthesis. To investigate the possibility that it was simply the nitrogen output which affected the enzyme levels, rats were fed gelatin instead of casein t o produce an increase in urinary nitrogen. In these experiments, although the urinary nitrogen did increase, the enzyme activities in the liver actually fell slightly. Body weight also appeared t o fall. In the converse experiment in which rats were changed from the diet with gelatin to the low protein diet, urinary nitrogen fell and the activity of the liver enzymes did not change. When nitrogen intake was related t o output in rats on the casein diet, there was an excellent linear correlation, with a slope indicating that a quantity of nitrogen was excreted in the urine equivalent t o half of the amount ingested. With gelatin there were fewer experiments, but there was still a linear correlation between urinary nitrogen and nitrogen intake. Therefore, this study shows that young rats can rapidly reduce their urinary nitrogen in response t o lowering dietary nitrogen, and within 30 hours a plateau i s reached. This provides new information, but it i s not as significant as the demonstration that a wide range of enzymes change in concert and a t about the same rate in response t o dietary manipulations. It i s striking that the enzymes measured differ in their intracellular location, and certainly
in the case of the urea cycle enzymes, differ also in basal activity. As the authors point out, the change in enzyme activity cannot be a reflection simply of an increase in urea output since increasing urea output by feeding gelatin did not cause a rise in the enzyme activity. The fact that urea excretion did change without a change in urea cycle enzymes is taken t o indicate that the "potential capacity" of the enzymes i s not fully utilized. Caution should be expressed here. The conditions under which the enzyme i s assayed in vitro obviously may have little relationship t o i t s activity in vivo since levels of such factors as substrate, co-factors, and pH will be different. I n those experiments in which the enzyme activities increase, could the effect be a nonspecific one of increased protein intake enhancing protein synthesis generally? In that case several other enzymes should show increases in activity. However, a nonspecific rise in soluble protein appears unlikely. With a change in dietary casein from 40 t o 135 g per kilogram, the total hepatic arginase activity rose some fourfold and aspartate aminotransferase rose similarly. It is unlikely that soluble liver protein wou Id increase four-fold. The authors considered the possibility that hormones might be the signal for the change in enzyme activity and referred t o the work of McLean and Gurney6 which showed that cortisol increased urea cycle enzymes. Here one should point out that in adrenalectomized rats those authors found that although the urea cycle enzymes changed, their degree of change was variable, and the aspartate aminotransferase activity did not change. S ~ h i m k e , how~ ever, has proven that steriods are not the signal for the changes in urea cycle enzymes in response to dietary alterations. He showed convincingly that the enzyme proteins in the two dietary states are identical and have identical kinetic characteristics. He also showed that the change in activity i s not simply due to presence of activators or absence of inhibitors. There is simply more enzyme protein. A fundamental difference between Schimke's work and the NUTRITION REVIEWWVOL. 33.
NO. 6 / JUNE
1975
181
present one is that the enzyme half-lives are so widely different. Schimke showed a half-life for arginase of about five days,8 as compared with eight hours in the present study. There obviously cannot be such a difference in enzyme protein in the two strains of rats. The reason for these differences is not clear, but perhaps relates to the different methods used, especially for calculations of enzyme half-lives. It must be confessed that there is no clue from this or other studies as to why these enzymes should change in concert. It is hard to believe that there is a single genome for all six enzymes. The pathway for which the single genome theory was proposed with most vigor i s gluc~neogenesis.~We know now that although some enzymes with widely differing specific activities do change together in response to some stimuli, there are some in the pathway which do not change, and the degree of change of those that do can be widely varied. It is hoped that the authors will pursue these studies, perhaps measuring the levels of the key intermediates t o determine if and how they do change, and measuring other enzymes as well. (Their data on glutamate dehydrogenase are in conflict with other studies.) The problem of the dietary control of enzyme synthesis and degradation is fundamental in a general physiological sense. We should know what signals the shunting of amino acids into the synthetic pathway and not into the pathway for degradation and/or excretion as a product which i s normally not reutilizable to any great extent. In situations when protein is present in short supply the form of control could be important for therapeutic reasons. There may be conditions such as
182
NUTRITION REVIEWSIVOL. 33, NO. 6 I JUNE 1975
after surgery or during hyperalimentation in which it may be desirable to conserve nitrogen by reducing the amount entering the pathway for degradation. It is not too naive to suggest that if we knew the mechanism of control it might be possible to adjust it. 0 1. J. C. Waterlow: The Partition of Nitrogen in the Urine of Malnourished Jamaican Infants. Am. J. Clin. Nutrition 12: 235-240, 1963 2. J. M. L. Stephen and J. C. Waterlow: Effect of Malnutrition on Activity of Two Enzymes Concerned with Amino Acid Metabolism in Human Liver. Lancet I: 118-119, 1968 3. N. S. Scrimshaw, M. A. Hussein, E. Murray, W. M. Raud, and V. R. Young: Protein Requirements of Man: Variations in Obligatory Urinary and Fecal Nitrogen Losses in Young Men. J. Nutrition 102: 1595-1604, 1972 4. H. Chan: Adaptation of Urinary Nitrogen Excretion in Infants to Changes in Protein Intake. Brit. J. Nutrition 22: 315-323, 1968 5. T. K. Das and J. C. Waterlow: The Rate of A d a p t a t i o n o f Urea Cycle Enzymes, Aminotransferases and Glutamate Dehydrogenase to Changes in Dietary Protein Intake. Brit. J. Nutrition 32: 353-373, 1974 6. P. McLean and M. W. Gurney: Effect of Adrenalectomy and of Growth Hormones on Enzymes Concerned with Urea Synthesis in R a t Liver. Biochem J. 87: 96-104, 1963 7. R. T. Schimke: Adaptive Characteristics of Urea Cycle Enzymes in the Rat. J. Biol. Chem. 237: 459-468, 1962 8. R . T. Schimke: The importance of Both Synthesis and Degradation in the Control of Arginase Levels in R a t Liver. J. Biol. Chem. 239: 3808-3817, 1964 9. G. Weber, R. L. Singhal, N. B. Stamm, E. A. Fisher, and M. A. Mentendiek: Regulation of Enzymes Involved in Gluconeogenesis. Adv. Enzyme Reg. 2: 1-38, 1964
