J. Nutr. Sci. Vitaminol., 26, 617-627, 1980 REGULATION OF TRYPTOPHAN-NIACIN METABOLISM BY HORMONES1 Hiroo SANADA and Motoyoshi MIYAZAKI2 Division of Basic Nutrition, National Institute of Nutrition, 1 Toyamacho, Shinjuku-ku, Tokyo 162, Japan (Received September 2, 1980) Summary The regulation of tryptophan-niacin metabolism by pi tuitary and adrenocortical hormones was investigated. Hypophysectomized rats fed on a niacin-free purified diet were injected with bovine somatotropin, predonine acetate or both. The urinary excretion of N-methylnicotinamide (MNA) and N-methyl-2-pyridone-5 - carboxamide (2-Py) after oral administration of tryptophan was then compared before and after the hormone treatment. The amount of urinary MNA was found to be increased after the injection of somatotropin, but decreased after the predonine injection. On the other hand, no change in urinary MNA was observed in the rats administered with both hormones. The amount of 2-Py appeared to be reduced by the predonine treatment, but was not affected by somatotropin injection. The activity of liver ƒ -amino-ƒà-carboxymuconate-ƒã-semialdehyde decarbo xylase (picolinic carboxylase) was shown to be directly proportional to the ratio (oral tryptophan)/(urinary MNA). Moreover, the enzyme activity appeared to be inversely proportional to the concentration of niacin in liver. In addition, the amount of urinary MNA was suggested to be affected by the change of body weight gain. The administration of both pituitary hormone and insulin failed to normalize the reduced urinary MNA excretion in diabetic rats, suggesting that any change in pituitary hormone was not responsible for the abnormal tryptophan-niacin metabolism in diabetes. Keywords tryptophan, niacin, N-methylnicotinamide, ƒ -amino-ƒàcarboxymuconate-ƒã-semialdehyde decarboxylase, hypophysectomized rat, somatotropin, predonisolone, diabetes, N-methyl-2-pyridone-5 - carboxamide It was found in our previous experiments that the ratio of oral tryptophan to urinary N-methylnicotinamide (MNA) was roughly proportional to the activity of liver ƒ -amino-ƒà-carboxymuconate-ƒã-semialdehyde decarboxylase [picolinic carbo- 1 Regulation of Tryptophan -Niacin Metabolism in Vivo (Part 3).
618 H. SANADA and M. MIYAZAKI xylase; EC 4.1 1,45] in diabetic rats (1). These findings suggest that the conversion rate of tryptophan to niacin in vivo is mainly regulated by this enzyme. Since niacin derivatives were confirmed to be biosynthesized from quinolinate, decarboxylation of ƒ -amino-ƒà-carboxymuconate-ƒã-semialdehyde, a precursor of quinolinate, by picolinic carboxylase has been considered as causing the reduction of quinolinate and niacin biosynthesis in vivo (2-4). In hypophysectomized rats, the activity of liver picolinic carboxylase was observed to be affected not only by adrenocortical hormone (5), but also by pituitary hormones such as somatotropin and prolactin (our previous paper). The present experiments were then performed to investigate the effect of somatotropin and adrenocortical hormones on the tryptophan-niacin metabolism in vivo. Moreover, we discussed the abnormal tryptophan-niacin metabolism in diabetic rats in relation to these hormones. MATERIALS AND METHODS Bovine somatotropin was obtained from Miles Laboratories, Inc. (Illinois, USA). Predonine acetate (predonisolone acetate for injection) was purchased from Shionogi Co. (Osaka, Japan). Methylnicotinamide, 3-hydroanthranilic acid, L tryptophan were obrained from Wako Junyaku Co. (Osaka, Japan). N-Methyl-2 - pyridone-5-carboxamide (2-Py) was the kind gift of Dr. T. Takahashi (Tsukuba University, Ibaraki-ken, Japan). Male Sprague Dawley rats hypophysectomized at 4 weeks of age were purchased from Clea Japan, Inc. (Tokyo, Japan). The animals were housed in individual metabolic cages settled in an air-conditioned room with a 12-hr lights on-of schedule. When they were 5 weeks old, a niacin-free experimental diet was given to them ad libitum, the composition of which is shown in Table 1. After 1 week, their urine was collected for 48 hr in a flask containing a small amount of toluene. When the first urine collection ended, L-tryptophan (30mg/rat) suspended in 0.01M acetic acid (15mg/ml) was administered orally by stomach tube and urine was collected again for the next 48hr. They were then divided into three groups of 5 rats each and administered with somatotropin (1mg/rat/ 12hr), predonine acetate (2.5mg/rat/ 12hr) or both 4 times every 12hr. The first group was injected with predonine acetate suspension (10mg/ml) intraperitoneally and the third group was given somatotropin solution (5mg/ml saline) subcutaneously. The second group was administered with somatotropin 1hr before predonine injection in the same J. Nutr. Sci. Vitaminol.
TRYPTOPHAN-NIACIN METABOLISM AND HORMONES 619 Table 1. Composition of the niacin-free experimental diet. * Composition of salt mixture and vitamin mixture was the same as described in our previous paper (1). way as other with groups. Thirty-six hr after the last injection of hormone, L tryptophan was administered again to all rats in the same manner as before. Then, urine was collected a third time over 48hr. Immediately after the completion of the urine collection, the rats were sacrificed by decapitation. The livers, were removed and chilled on ice as soon as possible. The diluted urine samples were filtered and stored at 20 Ž until analysis for MNA and 2-Py. The amounts of these urinary metabolites were determined by the methods of Takahashi (personal communication) partially modified by the authors. These methods were based on the report described by Price et al. (6). For the separation and purification of these metabolites, each urine sample was passed through triple-layered columns. Ion-exchange resin Dowex 1 and Dowex 50 were packed in the upper and middle columns, respectively. The bottom column contained the mixture of cellulose and charcoal (Nucher C-190N, Wako Junyaku Co.). After the washing of these columns, MNA was eluted from the Dowex 50 column by 1M sodium chloride solution containing 0.1N hydrochloride, and 2-Py was recovered from the charcoal column by washing with 50% ethanol solution containing 2% pyridine. The eluate containing 2-Py was evaporated to dryness. Then the residue dissolved in water was passed through a double-layered column. The upper and lower columns were packed with Dowex 1 and Dowex 50, respectively. 2-Py was then washed out from the Dowex 50 column by water. Thereafter, the 2-Py solution was washed with ethyl acetate and ethyl ether (both saturated with water) and the concentration of 2-Py was determined photometri cally at 258nm. The concentration of MNA was determined by the method of Price (6). The activity of picolinic carboxylase was assayed as described previously (1). The amount of nicotinate in liver was determined microbiologically after the sample was hydrolyzed in 1 N sulfuric acid (1). Vol. 26, No. 6, 1980
620 H. SANADA and M. MIYAZAKI RESULTS The body weight gains of hypophysectomized rats were significantly increased by the injection of bovine somatotropin (Fig. 1). The administration of predonine, however, decreased body weight. Somatotropin was shown to partially protect the hypophysectomized rats from weight loss caused by predonine. The food intake of the rats treated with somatotropin or predonine was greater than that before injection of hormone (Fig. 2). However, the rats administered-with both somatotropin and predonine consumed about the same amount of diet as before hormone treatment. The amount of MNA excreted in urine for 48 hr is indicated in Fig. 3(A). Before the hormone treatment, oral administration of L-tryptophan increased the amount of urinary MNA significantly. When the hypophysectomized rats were loaded with L-tryptophan after somatotropin injection, the amount of urinary MNA was found to be significantly increased. The administration of predonine, on the contrary, reduced the amount of MNA. Whereas, in the rats given both somatotropin and predonine, the urinary exretion of MNA was observed to be unchanged. The amount of 2-Py excreted in urine is shown in Fig. 3(B). Injection of predonine with or without somatotropin resulted in the reduction of urinary 2-Py levels. Somatotropin, on the other hand, was shown to have no significant effect on the excretion of 2-Py. These results are considered to indicate that the hormonal regulation of MNA and 2-Py production differ from each other. Fig. 1. Body weight before and after hormone treatment. Hypophysectomized rats were injected with predonine ( ~), somatotropin ( ) or both ( ) 4 times every 12 hr (at the time indicated by arrows). The periods of urine collection are indicated by shaded bar. Fig. 2. Food intake before and after hormone treatment. Food intake of each group was measured in the periods of urine collection. _??_, + predonine; _??_, + predonine +somatotropin; _??_, + somatotropin. J. Nutr. Sci, Vitnminol.
TRYPTOPHAN-NIACIN METABOLISM AND HORMONES 621 Fig. 3. Urinary excretion of N-methylnicotinamide and N-methyl-2-pyridone-5 -carboxamide. A, N-methylnicotinamide; B, N-methyl-2-pyridone-5-carboxamide. _??_, + predonine; _??_, + predonine + somatotropin; _??_, + somatotropin. Table 2. Concentration of total niacin in liver. Values represent mean +S.E.M. Means in the same column not sharing a common superscript letter differ significantly (p<0.05). After the hydrolysis of liver samples, the concentration of nicotinate in liver was analyzed (Table 2). The administration of somatotropin alone appeared to increase the niacin content of liver. As the niacin content of normal rat liver is about 1.69 }0.02ƒÊmol/g liver (n=5), that of the hypophysectomized rat treated by somatotropin was thought to be slightly higher. The activity of liver picolinic carboxylase was measured immediately after the third urine collection (84hr after the last injection of hormone) (Table 3). The enzyme activity of the group injected with somatotropin alone was observed to be significantly lower than that of the group given predonine with or without somatotropin. These results appeared to indicate that somatotropin could not suppress the enzyme induction by predonine. We then tried to estimate the enzyme activity at the beginning of the third urine collection (36hr after the last hormone Vol. 26, No. 6, 1980
622 H. SANADA and M. MIYAZAKI Table 3. The activity of liver picolinic carboxylase. Values represent mean }S. E. M. Means in the same column not sharing a common superscript letter differ significantly (p<0.05). Fig. 4. Time course of the activity of liver picolinic carboxylase after the injection of predonine and somatotropin. A, tryptophan pyrrolase; B, picolinic carboxylase. 0, + somatotropin;, + somatotropin + predonine; ~, + predonine. injection). For this purpose, the same experimental program of hormone treatment as described above was repeated again using other hypophysectomized rats. Twelve hr after the last injection of hormone, the activity of liver picolinic carboxylase was found to be significantly low in the rats which had received both somatotropin and predonine (Fig. 4). However, the enzyme activity of this group was gradually elevated to reach the same level as that shown with predonine alone 84 hr after the last injection of hormone. The ratio of the enzyme activity at 36 hr to that at 84 hr in this group was calculated to be 0.61 and in the case of predonine alone, 1. 12. J. Nutr. Sci. Vitaminol.
TRYPTOPHAN-NIACIN METABOLISM AND HORMONES 623 Therefore, the enzyme activities at the begining of the third urine collection in the former experiments (36hr after the last hormone injection) were estimated by multiplying these ratios into the activities at 84hr (Table 3). In the rats administered with somatotropin alone, it was considered that the enzyme activity 36 hr after the last injection of hormone was the same low level as that at 84hr. The activity of liver tryptophan pyrrolase was also measured in the experiments of time-course of picolinic carboxylase (Fig. 4). The induction of tryptophan pyrrolase by predonine was not shown to be suppressed by the administration of somatotropin. Therefore, in the two predonine-treated groups, this enzyme is likely to have an effect on the tryptophan-niacin metabolism. Since liver picolinic carboxylase was thought to be one of the most important enzymes for controlling the conversion rate of tryptophan to niacin, the relationship between the ratio of oral tryptophan to urinary MNA and the activity of liver picolinic carboxylase was investigated. As shown in Fig. 5, the elevation of Fig. 5. Correlation between the activity of liver picolinic carboxylase and urinary N - methylnicotinamide excretion. Estimated activities of liver picolinic carboxylase were the same as those shown in Table 3. The ratios of oral tryptophan to urinary MNA in the third urine-collection period are indicated on the abscissa. The amount of oral tryptophan is the sum of the tryptophan administered and in the diet. ~, + predonine;, + somatotropin + predonine;, + somatotropin. Fig. 6. Effect of rat pituitary extract and insulin on the urinary excretion of MNA in diabetic rats. About 80 days after the alloxan injection, three diabetic rats were administered with both rat pituitary extract (equivalent to 4 hypophyses per rat) and insulin (5 U per rat) every 12hr for 7 days. All rats were fed on a niacin-free diet as shown in Table 1, Their urine was collected 1 day before hormone treatment and on the last day of the hormone injection period., nondiabetic control; œ, diabetic. Vol. 26, No. 6, 1980
624 H. SANADA and M. MIYAZAKI the enzyme activity was found to be accompanied by a greater value of the ratio. This observation indicates that the amount of tryptophan required to produce a constant amount of MNA increases according to the elevation of the activity of liver picolinic carboxylase. Next, experiments were performed to investigate the relationship between the regulatory function of pituitary hormone and the abnormal tryptophan-niacin metabolism in diabetic rats. When rat pituitary extract was administered with insulin to the diabetic rats for 7 days every 12hr, urinary excretion of MNA per food intake was rather reduced and failed to normalize (Fig. 6). These results suggest that the decreased amount of MNA in diabetic rats is not caused by changes in levels of their pituitary hormones. DISCUSSION In the present report, tryptophan-niacin metabolism was clearly shown to be affected by pituitary and adrenocortical hormones. The amount of urinary MNA in hypophysectomized rat was found to be increased by the administration of somatotropin. On the other hand, 2-Py levels were not increased by the same hormone. Whereas, predonine reduced the urinary excretion of both MNA and 2 - Py. These results indicate that the regulation of MNA production is probably different from that of 2-Py. In the diabetic rats, the change of MNA excretion has been shown to be roughly inversely proportional to the activity of liver picolinic carboxylase (1, 7). Thus, it is considered to be reasonable that the change of the molar ratio (oral tryptophan)/(urinary MNA) is proportional to the activity of liver picolinic carboxylase in hypophysectomized rats. Greengard reported that the concentration of liver NAD was increased in hypophysectomized rats and that adrenalectomy was effective in elevating NAD in liver (8). Moreover, the injection of adrenocorti cal hormone to the niacin-deficient rats has been shown to prevent the decrease of liver NAD and NADH (9). As the adrenocortical hormone was well known to have a catabolic action on tissue protein, tryptophan-niacin metabolism in predonine treated rats was considered to be affected by the release of tryptophan and niacin from tissue. Although the differences in the ratio (oral tryptophan)/(urinary MNA) among these three groups were reasonably explained by the changes of liver picolinic carboxylase levels, small variations of these ratios in each group were likely to be affected by the retention or release of tryptophan and niacin in tissue. For this reason, we assumed that a constant amount of tryptophan per g of body weight change had to be added or subtracted from oral tryptophan intake. The hypothetical equation is shown as follows: (1) where a and b are unknown coefficients. The velocity of spontaneous quinolinate J. Nutr, Sci. Vitaminol.
TRYPTOPHAN-NIACIN METABOLISM AND HORMONES 625 production and that of the decarboxylation of a-amino-fl-carboxymuconate-e semialdehyde by picolinic carboxylase (activity of picolinic carboxylase in vivo) are expressed by V1 and V2, respectively (1). The change of body weight is expressed by dw. After Eq. (1) was rewritten into another form, correlation between the values of (urinary MNA) (1+nVo)/ w and (oral tryptophan)/ w was examined (Fig. 7), where Vo and n means the activity of picolinic carboxylase in vitro and its coefficient, respectively, which can be estimated from Fig. 5 if the effect of body weight change is neglected. The above two values are in direct proportion and the following equation is obtained from Fig. 7, (urinary MNA)=0.031{(oral tryptophan)-40 w}{1+1.5(pcase)}-1 where PCase means the activity of liver picolinic carboxylase; the amounts of urinary MNA and oral tryptophan must be expressed inĐmoles. This equation suggests that 1g of weight loss causes the release of tissue tryptophan and niacin Fig. 7. The effect of body weight gain on the excretion of N-methylnicotinamide. Hypothetical equation (explained in DISCUSSION) Rewritten form The value of n can be estimated from Fig. 5 if the effect of body weight gain is neglected as follows: when b w=0, hypothetical equation is (urinary MNA)=a(oral tryptophan){1+n(pcase)}-1 when the ratio (oral tryptophan)/(urinary MNA) in Fig. 5 is 0, (PCase)=-n-1 Vol. 26, No. 6, 1980
626 H. SANADA and M. MIYAZAKI Fig. 8. Correletion between the concentration of niacin in liver and the activity of liver picolinic carboxylase. ~, + predonine;, + somatotropin + predonine;, + somatotropin. The concentration of niacin in normal liver is indicated by shaded area. equivalent to 40ƒÊmol of tryptophan. The niacin content of the rat liver was found to be roughly inversely proportional to the activity of picolinic carboxylase (Fig. 8). This observation also suggests the importance of the enzyme in the tryptophan-niacin metabolism. However, such correlation may not be observed in the normal rat, because the half life of niacin was reported to be significantly longer in hypophysectomized rats than in normal animals (10, 11). Since the reduced MNA excretion in diabetic rats was not normalized by the administration of both pituitary extract and insulin, changes in pituitary hormone levels were not considered to be responsible for the abnormality of tryptophan niacin metabolism. The authors wish to thank Drs. Tetsuzo Takahashi (The University of Tsukuba, Ibaraki-ken, Japan) and Takeshi Suzuki (National Institute of Public Health, Tokyo, Japan) for their helpful advice and discussions. We are also grateful to Dr. Tatsuo Abe (Medical School, Toho University, Tokyo, Japan) for his critical reading of the manuscript and valuable suggestions. REFERENCES 1) Sanada, H., Miyazaki, M., and Takahashi, T. (1980): Regulation of tryptophan-niacin metabolism in diabetic rats. J. Nutr. Sci. Vitaminol., 26, 449-459. 2) Ikeda, M., Tsuji, H., Nakamura, S., Ichiyama, A., Nishizuka, Y., and Hayaishi, 0. (1965): Studies on the biosynthesis of nicotinamide adenine dinucleotide. II. A role of picolinic carboxylase in the biosynthesis of nicotinamide adenine dinucleotide from tryptophan in mammals. J. Biol. Chem., 240, 1395-1401. 3) Mehler, A. H., Yano, K., and May, E. L. (1964): Nicotinic acid biosynthesis: Control by an enzyme that compete with a spontaneous reaction. Science, 145, 817-819. J. Nutr. Sci. Vitaminol.
TRYPTOPHAN-NIACIN METABOLISM AND HORMONES 627 4) Nishizuka, Y., and Hayaishi, O. (1971): Picolinic carboxylase in reference to NAD biosynthesis, in Methods in Enzymology, ed. by Colowick, S. P. and Kaplan, N. O., Academic Press, New York, Vol. 18, pp. 162-175. 5) Mehler, A. H., McDaniel, E. G., and Hundley, J. M. (1957): Changes in the enzymatic composition of liver. II. Influence of hormones on picolinic carboxylase and tryptophan peroxidase. J. Biol. Chem., 232, 331-335. 6) Price, J. M., Brown, R. R., and Yess, N. (1965): Testing the functional capacity of the tryptophan-niacin pathway in man by analysis of urinary metabolites, in Advances in Metabolic Disorders, ed. by Levine, R., and Luft, R., Academic Press, New York, pp. 159-225. 7) Mehler, A. H., McDaniel, E. G., and Hundley, J. M. (1957): Changes in the enzymatic composition of liver. I. Increase of picolinic carboxylase in diabetes. J. Biol. Chem., 232, 323-330. 8) Greengard, P., Quinn, G. P., and Reid, M. B. (1965): Identification of hormones affecting pyridine nucleotide metabolism of rat liver. J. Biol. Chem., 240, 486-490. 9) Greengard, P., Kalinsky, H., Manning, T. J., and Zak, S. B. (1968): Prevention and remission by adrenocortical steroids of nicotinamide deficiency disease. II. A study of the mechanism. J. Biol. Chem., 243, 4216-4221. 10) Greengard, P., Quinn, G. P., and Reid, M. B. (1964): Pituitary influence on pyridine nucleotide metabolism of rat liver. J. Biol. Chem., 239, 1887-1892. 11) Greengard, P., Petrack, B., and Kalinsky, H. (1967): Effect of hypophysectomy on pyridine nucleotide metabolism. J. Biol. Chem., 242, 152-154. Vol. 26, No. 6, 1980