- Email: [email protected]
Stimulation of two adaptive rat liver enzymes by quinolinic acid as a function of time
iâ
Pri~éd
eat Hritaiâl'
9. Part II, pp, 901-905, 1970 .
Pergamon Press
STIMULATION OF TWO ADAPTIVE RAT LIVER BNZYMES BY QUINOLINIC ACID AS A FUNCTION OF TI1IS Rüdiger Hardeland I . Zoologisches Institut Universität Göttingen bleat Germany (Received 3 Aprll 1970 ; in final form 11 June 1970) BOTH tryptophan pyrrolase (L-tryptophan :02 ozidoreductase ; >~ 1 . 13 .1 .12 .) and tyrosine transaminaee (L-tyrosine :2-ozoglutarate aminotransferaee ; S+C 2 .6 .1 .5 .) have been shown to be adaptive in response to various compounds : tryptophan pyrrolase is induced by its substrate, tryptophan (1,2,3), and some of its analogues (4) as well as by hydrocortisone (5,6) and, to a lesser extent, by insulin (7) ; tyrosine transaminaae oan be induced by tyrosine (8), tryptophan (4), pyridoxine (9), hydrocortisone (5,6,10), glucagon (11,12), insulin (12), epinephrine (13), and 3',5'cyclic AMP (13) " It is remarkable that all hormones which are known to elevate tryptophan pyrrolase and tyrosine traneaminase levels are powerful regulators of the carbohydrate metabolism . This report is concerned with the effects of quinolinio acid on the activities of both enzymes . Quinolinic acid is a tryptophan metabolite which inhibits gluconeogeneais (14) . Materiala ând Methods Male Sprague-Dawley rats, 3 - 5 months old and weighing about 300 g, were kept under a light-dark cycle of 12 :12 h . Since it is known that tyrosine transaminaee as well as tryptophan pyr901
902
II~UCTION OF ENZYME BY QUINOLINIC ACm Vo1 .9, No .18
rolase activities vary within the 24-hour period (15,16,17), all animals were sacrificed at the same time of day . Injections were given intraperitoneally at a dose of 25 mg quinolinic acid per 100 g body weight, in varying intervals between the times of injection and of sacrificing the rata . Liver pieces of about 1 g from the left posterior lobe were homogenized in 0 .14 M KC1 containing 0 .0025 N NaOH . The homogenates were centrifuged at~20,000g for 20 min. The enzyme assay miature for tryptophan pyrrolase was essentially that described by FeYgeleon and Greèngard (18) : 1 .0 ml of supernate, 1 .4 ml of potassium phosphate buffer 0 .2 M, pH 7 .0, 0 .3 ml of L-tryptophan 0 .03 ff, 0 .3 ml of hemin 0 .002 mM . Tyrosine transaminase activity was assayed by the method of Diamondatone (19) . kesulta Several hours after injection of quinolinic acid, both tryptophan pyrrolase and tyrosine transaminase show an adaptive increaae in activity, the latter to a greater extent than the for mer (fig . 1) . In either case, 6 hours was sufficient to obtain maximal or nearly maximal values . A statistical discrimination between the tryptophan pyrrolase values for 4, 6 and 8 hours and between the tyrosine transaminase values for 6, 8 and 10 hours was not possible . Nonetheless, the rise in both enzyme activities was highly significant (cf . 3fold S .S .) . One day after injection, the original tryptophan pyrrolase level was restored, whereas tyrosine transaminase activity had declined to subnormal values . Discussion A known effect of quinolinic acid is tt :e in viva inhibition of phosphoenolpyruvate carboxykinase, which results in an elevation of hepatic pyruvate, lactate, citrate, malate, aspartate and
Vol . 9, No . 18
IIVDUCTION OF ENZYME BY QUINOLINIC AC1D
r 13001
13 ,~
> 1200-
12
~1100Y ~ Q 1000-~
11~
~n
9~ w
~ ~ 800-i Ca
8
C ~ 6001
6~
d X ~rQ ~a ~a
903
5~
Y O E
g ____ _j 91
_c ô
sL g_ ~~~9 0
2
4
6 8 10 24 hours aRsr injsction FIG .
4 3 ~. a 2 ,ô 1 ~
28
1
Increase in tryptophan pyrrolase and tyrosine traneaminase activities in response to quinolinic acid . Dashed lineal tryptophan pyrrolase ; solid 11nes : tyrosine traneaminase ; vertical lineal 3iold standard error; the numbers indicate the number of animals used per point . oxalacetate concentrations and a decrease in the levels oß phosphoenolpyruvate, 2- and 3-phosphoglyceric acids and hexosemonophosphatea (14,20) . Since no direct activation by quinolinic acid of tyrosine transaminase or tryptophan pyrrolase has been observed (unpublished data), it is moat likely that the stimulation of both enzyme activities is due to either the accumulation or the depletion of certain carbohydrate metabolites . One way of action may involve a hormonal counter-regulation, because the inhibition
904
II~UCTiON OF ENZYME HY QUINOLINIC ACID
Vol, 9, No . 18
oß phoaphoenolpyruvate carboxykinase results in hypoglycemia, which in turn leads to supernormal secretion of enzyme-inducing hormones such as glucocorticoida and, in the case of tyrosine transaminase, glucagon . However, some preliminary eaperimenta with liver slices (unpublished) indicate that a control mechanism independent oß blood glucose levels is also aßfected by the treatment with quinolinic acid . This work was supported by the Deutsche Forachungagemeinschaft .
References 1 .
W . S. KNOX and A . H . MEHLER, Science 1~, 237 (1951) .
2.
0 . GREENGARD and F . FEIGELSON, J . Biol . Chem . ~, 158 (1961) .
3.
P . FEIGELSON and 0 . GREENGARD, J . Biol . Chem . 2~, 1908 (1962) .
4.
F . ROSEN and R . J . MILHOLLAND, J . Biol . Chem . ~_, 3730 (1963) .
5.
L . GOLDSTEIN, E . J . STELLA and W . E . KNOX, J . Biol . Chem . 1723 (1962) .
6.
0 . GREENGARD, G . T . BAKER, ~S . L . HOROWITZ and W . E . KNOX, Proc . Nat . Aoad . Sci . (Wash~ ~, 1303 (1966) .
7.
J . M . SCHOR and E . FRIEDEN, J . .Biol . Chem . 23~, 612 (1958) "
8.
B . C . C . LIN and W . E . KNOX, J . Biol . Chem . ~, 1186 (1958) "
9.
D . HOLTEN, W . D . WIC1iS and F . T . KENNEY, J . Biol . Chem . 2~2, 1053 (1967) .
10 .
E . C . C . LIN and W . E . KNOX, Biochim . Biovhys . Acta 26, 85 (1957) .
11 .
0 . GREENGARD and H . K . DENEY, J . Biol . Chsm . ~, 2986 (1967) .
12 .
D . HOLTEN and F . T . KENIdEY, J . Biol . Chem . ~, 4372 (1967) .
13 .
W . D . WICF:S, Science 160, 997 (1968) .
14 .
C . h1 . VEIv'EZIALE, P . WALTER, N . KNEER and H. ~. . LARDY, Biochemistry 6, 2129 (1967) .
15 .
R . J . WURT~TAN and J . AXELROD, Proc . Nat . kcad . Sci . (Hash .) 51, 1594 (1967 ) .
Vol . 9, No . 16
II~UCTION OF ENZYME BY QUINOLINIC ACID
905
16 .
M . CIVEN, R . ULRICH, B . M. TRIhIMER and C . B . BROWN, Science 1563 (1967) .
17 .
R . HARDTLAND and L. REN3ING, Nature 21~, 619 (1968) .
18 .
P. FEIGEZ30N and 0 . GR>3ENGARD, J . Biol . Chem . ~, 153 (1961) .
19 .
T . I . DIAèHOND3TONT, Analst .,Biochem .
20 .
H . A . LARDY, Inhibitors Toole in Cell Research, p . 374 . Sd . Th . Bücher, ea, pr nger- er ag, er n - Heidelberg New York 1969 .
6, 395 (1966) .
Pri~éd
eat Hritaiâl'
9. Part II, pp, 901-905, 1970 .
Pergamon Press
STIMULATION OF TWO ADAPTIVE RAT LIVER BNZYMES BY QUINOLINIC ACID AS A FUNCTION OF TI1IS Rüdiger Hardeland I . Zoologisches Institut Universität Göttingen bleat Germany (Received 3 Aprll 1970 ; in final form 11 June 1970) BOTH tryptophan pyrrolase (L-tryptophan :02 ozidoreductase ; >~ 1 . 13 .1 .12 .) and tyrosine transaminaee (L-tyrosine :2-ozoglutarate aminotransferaee ; S+C 2 .6 .1 .5 .) have been shown to be adaptive in response to various compounds : tryptophan pyrrolase is induced by its substrate, tryptophan (1,2,3), and some of its analogues (4) as well as by hydrocortisone (5,6) and, to a lesser extent, by insulin (7) ; tyrosine transaminaae oan be induced by tyrosine (8), tryptophan (4), pyridoxine (9), hydrocortisone (5,6,10), glucagon (11,12), insulin (12), epinephrine (13), and 3',5'cyclic AMP (13) " It is remarkable that all hormones which are known to elevate tryptophan pyrrolase and tyrosine traneaminase levels are powerful regulators of the carbohydrate metabolism . This report is concerned with the effects of quinolinio acid on the activities of both enzymes . Quinolinic acid is a tryptophan metabolite which inhibits gluconeogeneais (14) . Materiala ând Methods Male Sprague-Dawley rats, 3 - 5 months old and weighing about 300 g, were kept under a light-dark cycle of 12 :12 h . Since it is known that tyrosine transaminaee as well as tryptophan pyr901
902
II~UCTION OF ENZYME BY QUINOLINIC ACm Vo1 .9, No .18
rolase activities vary within the 24-hour period (15,16,17), all animals were sacrificed at the same time of day . Injections were given intraperitoneally at a dose of 25 mg quinolinic acid per 100 g body weight, in varying intervals between the times of injection and of sacrificing the rata . Liver pieces of about 1 g from the left posterior lobe were homogenized in 0 .14 M KC1 containing 0 .0025 N NaOH . The homogenates were centrifuged at~20,000g for 20 min. The enzyme assay miature for tryptophan pyrrolase was essentially that described by FeYgeleon and Greèngard (18) : 1 .0 ml of supernate, 1 .4 ml of potassium phosphate buffer 0 .2 M, pH 7 .0, 0 .3 ml of L-tryptophan 0 .03 ff, 0 .3 ml of hemin 0 .002 mM . Tyrosine transaminase activity was assayed by the method of Diamondatone (19) . kesulta Several hours after injection of quinolinic acid, both tryptophan pyrrolase and tyrosine transaminase show an adaptive increaae in activity, the latter to a greater extent than the for mer (fig . 1) . In either case, 6 hours was sufficient to obtain maximal or nearly maximal values . A statistical discrimination between the tryptophan pyrrolase values for 4, 6 and 8 hours and between the tyrosine transaminase values for 6, 8 and 10 hours was not possible . Nonetheless, the rise in both enzyme activities was highly significant (cf . 3fold S .S .) . One day after injection, the original tryptophan pyrrolase level was restored, whereas tyrosine transaminase activity had declined to subnormal values . Discussion A known effect of quinolinic acid is tt :e in viva inhibition of phosphoenolpyruvate carboxykinase, which results in an elevation of hepatic pyruvate, lactate, citrate, malate, aspartate and
Vol . 9, No . 18
IIVDUCTION OF ENZYME BY QUINOLINIC AC1D
r 13001
13 ,~
> 1200-
12
~1100Y ~ Q 1000-~
11~
~n
9~ w
~ ~ 800-i Ca
8
C ~ 6001
6~
d X ~rQ ~a ~a
903
5~
Y O E
g ____ _j 91
_c ô
sL g_ ~~~9 0
2
4
6 8 10 24 hours aRsr injsction FIG .
4 3 ~. a 2 ,ô 1 ~
28
1
Increase in tryptophan pyrrolase and tyrosine traneaminase activities in response to quinolinic acid . Dashed lineal tryptophan pyrrolase ; solid 11nes : tyrosine traneaminase ; vertical lineal 3iold standard error; the numbers indicate the number of animals used per point . oxalacetate concentrations and a decrease in the levels oß phosphoenolpyruvate, 2- and 3-phosphoglyceric acids and hexosemonophosphatea (14,20) . Since no direct activation by quinolinic acid of tyrosine transaminase or tryptophan pyrrolase has been observed (unpublished data), it is moat likely that the stimulation of both enzyme activities is due to either the accumulation or the depletion of certain carbohydrate metabolites . One way of action may involve a hormonal counter-regulation, because the inhibition
904
II~UCTiON OF ENZYME HY QUINOLINIC ACID
Vol, 9, No . 18
oß phoaphoenolpyruvate carboxykinase results in hypoglycemia, which in turn leads to supernormal secretion of enzyme-inducing hormones such as glucocorticoida and, in the case of tyrosine transaminase, glucagon . However, some preliminary eaperimenta with liver slices (unpublished) indicate that a control mechanism independent oß blood glucose levels is also aßfected by the treatment with quinolinic acid . This work was supported by the Deutsche Forachungagemeinschaft .
References 1 .
W . S. KNOX and A . H . MEHLER, Science 1~, 237 (1951) .
2.
0 . GREENGARD and F . FEIGELSON, J . Biol . Chem . ~, 158 (1961) .
3.
P . FEIGELSON and 0 . GREENGARD, J . Biol . Chem . 2~, 1908 (1962) .
4.
F . ROSEN and R . J . MILHOLLAND, J . Biol . Chem . ~_, 3730 (1963) .
5.
L . GOLDSTEIN, E . J . STELLA and W . E . KNOX, J . Biol . Chem . 1723 (1962) .
6.
0 . GREENGARD, G . T . BAKER, ~S . L . HOROWITZ and W . E . KNOX, Proc . Nat . Aoad . Sci . (Wash~ ~, 1303 (1966) .
7.
J . M . SCHOR and E . FRIEDEN, J . .Biol . Chem . 23~, 612 (1958) "
8.
B . C . C . LIN and W . E . KNOX, J . Biol . Chem . ~, 1186 (1958) "
9.
D . HOLTEN, W . D . WIC1iS and F . T . KENNEY, J . Biol . Chem . 2~2, 1053 (1967) .
10 .
E . C . C . LIN and W . E . KNOX, Biochim . Biovhys . Acta 26, 85 (1957) .
11 .
0 . GREENGARD and H . K . DENEY, J . Biol . Chsm . ~, 2986 (1967) .
12 .
D . HOLTEN and F . T . KENIdEY, J . Biol . Chem . ~, 4372 (1967) .
13 .
W . D . WICF:S, Science 160, 997 (1968) .
14 .
C . h1 . VEIv'EZIALE, P . WALTER, N . KNEER and H. ~. . LARDY, Biochemistry 6, 2129 (1967) .
15 .
R . J . WURT~TAN and J . AXELROD, Proc . Nat . kcad . Sci . (Hash .) 51, 1594 (1967 ) .
Vol . 9, No . 16
II~UCTION OF ENZYME BY QUINOLINIC ACID
905
16 .
M . CIVEN, R . ULRICH, B . M. TRIhIMER and C . B . BROWN, Science 1563 (1967) .
17 .
R . HARDTLAND and L. REN3ING, Nature 21~, 619 (1968) .
18 .
P. FEIGEZ30N and 0 . GR>3ENGARD, J . Biol . Chem . ~, 153 (1961) .
19 .
T . I . DIAèHOND3TONT, Analst .,Biochem .
20 .
H . A . LARDY, Inhibitors Toole in Cell Research, p . 374 . Sd . Th . Bücher, ea, pr nger- er ag, er n - Heidelberg New York 1969 .
6, 395 (1966) .