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Additive stimulation of hepatic putrescine production by glucagon and insulin after partial hepatectomy in rats
Life Sciences, Vol. 45, pp. 77-83 Printed in the U.S.A.
ADDITIVE
S T I M U L A T I O N OF H E P A T I C P U T R E S C I N E P R O D U C T I O N BY G L U C A G O N AND INSULIN A F T E R P A R T I A L H E P A T E C T O M Y IN RATS Yuzuru
First
Pergamon Press
Sato, Kenji F u j i w a r a * , Itsuro Ogata, T o m o a k i T o m i y a and Hiroshi Oka
D e p a r t m e n t of M e d i c i n e , F a c u l t y of Medicine, U n i v e r s i t y Tokyo, 7-3-I Hongo, B u n k y o - k u , T o k y o 113, J a p a n
of
(Received in final form April 28, 1989) Summary W h e n rats r e c e i v e d g l u c a g o n or insulin every 2 h a f t e r p a r t i a l h e p a t e c t o m y (Hx), h e p a t i c p u t r e s c i n e c o n t e n t was i n c r e a s e d above control levels at 6 and 12 h, r e s p e c t i v e l y . W h e n the two h o r m o n e s were combined, the i n c r e a s e d levels were additive. Hepatic o r n i t h i n e d e c a r b o x y l a s e a c t i v i t y was above c o n t r o l l e v e l s at 12 h a f t e r i n s u l i n t r e a t m e n t . Hepatic spermidine N1-acetyltransferase activity was enh a n c e d at 6 h only w h e n g l u c a g o n was dosed. P u t r e s cine a d m i n i s t r a t i o n from 0 to 4 h or from 6 to 10 h i n c r e a s e d h e p a t i c DNA s y n t h e s i s to similar levels 22 h a f t e r Hx. These r e s u l t s suggest that g l u c a g o n and insulin additively stimulate hepatic putrescine p r o d u c t i o n after Hx. This may e x p l a i n the c o o p e r a tive s t i m u l a t i o n of liver r e g e n e r a t i o n by both hormones. It has been s u g g e s t e d that g l u c a g o n and insulin c o o p e r a t e in s t i m u l a t i n g liver r e g e n e r a t i o n (I-3). The m e c h a n i s m of s t i m u l a tion, however, is still unclear. It is w i d e l y a c c e p t e d that p o l y a m i n e s play an i m p o r t a n t role in cell d i v i s i o n (4). We have o b t a i n e d e v i d e n c e that p u t r e s c i n e is n e c e s s a r y for DNA s y n t h e s i s in liver r e g e n e r a t i o n after partial h e p a t e c t o m y in rats, and is s u p p l i e d not only via the route of c o n v e r s i o n of o r n i t h i n e into p u t r e s c i n e by o r n i t h i n e d e c a r b o x y l a s e (ODC) but also via the c o m p e n s a t o r y route of s p e r m i d i n e conversion into p u t r e s c i n e by spermidine N1-acetyltransferase (SAT) and p o l y a m i n e o x i d a s e (5). G l u c a g o n and insulin, on the o t h e r h a n d , m a y a c t on l i v e r r e g e n e r a t i o n at t h e G 1 - p h a s e of cell c y c l e (2). Thus, both h o r m o n e s m i g h t affect h e p a t i c p u t r e s cine p r o d u c t i o n a f t e r partial h e p a t e c t o m y . In the p r e s e n t paper, we s t u d i e d this p o s s i b i l i t y the m e c h a n i s m of s t i m u l a t i o n by both h o r m o n e s in rats.
*To w h o m
correspondence
should
be addressed.
0024-3205/89 $3.00 +.00 Copyright (c) 1989 Pergamon Press plc
to c l a r i f y
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Hormones on Liver Putrescine Production
Materials
Vol. 45, No. I, 1989
and M e t h o d s
Experiments Male Sprague-Dawley rats (Charles River Japan, Atsugi, Japan) w e i g h i n g 180-200 g w e r e fed a c o m m e r c i a l p e l l e t e d diet and w a t e r ad l i b i t u m in a c a g e at 2 2 ± 2 ° C u n d e r n o r m a l l a b o r a t o r y lighting conditions. B e t w e e n 9:00 and 11:00 am, they u n d e r w e n t t w o - t h i r d s r e s e c t i o n of the liver a c c o r d i n g to the m e t h o d of Higgins and A n d e r s o n (6) u n d e r light ether a n a e s t h e s i a , and w e r e g i v e n w a t e r ad libitum. E x p e r i m e n t I: H e p a t e c t o m i z e d rats were d i v i d e d into 4 groups. In 3 h o r m o n e - t r e a t e d groups, they r e c e i v e d a s u b c u t a n e o u s i n j e c t i o n of 5 m l / k g body wt of 5% g l u c o s e solution c o n t a i n i n g 5 u g / m l of g l u c a g o n (Novo Industri A/S, C o p e n h a gen, Denmark) a n d / o r 8 mU/ml of A c t r a p i d i n s u l i n (Novo) e v e r y 2 h i m m e d i a t e l y a f t e r o p e r a t i o n , until 2 h b e f o r e sacrifice. In one g r o u p (control), they r e c e i v e d a s i m i l a r dose of the g l u c o s e solution alone. At the time of sacrifice, under ether a n a e s t h e s i a , the liver was p e r f u s e d w i t h 12 ml of i c e - c o l d saline. Then the liver was excised, q u i c k l y f r o z e n in liquid nitrogen, and stored at -80°C until it was used for d e t e r m i n a t i o n s of h e p a t i c a c t i v i ties of O D C and SAT and p u t r e s c i n e content. E x p e r i m e n t II: Hep a t e c t o m i z e d rats were d i v i d e d into 4 groups. In 2 groups, they r e c e i v e d an ip i n j e c t i o n of 1 m l / k g body wt of 0.2 m m o l e / m l putrescine dihydrochloride (Wako Pure C h e m i c a l Industries Ltd., Osaka, Japan) s o l u t i o n in saline a d j u s t e d to pH 7.4 by a d d i t i o n of N a O H at 0, 2 and 4 h or at 6, 8 and 10 h after o p e r a t i o n , and in the other 2 groups, they r e c e i v e d the same v o l u m e of saline alone similarly. They were k i l l e d at 12 h for the d e t e r m i n a t i o n of h e p a t i c p u t r e s c i n e content, as above. In a n o t h e r 4 g r o u p s of h e p a t e c t o m i z e d rats f o l l o w e d by t r e a t m e n t with p u t r e s c i n e dihyr o c b l o r i d e or saline, they r e c e i v e d an ip i n j e c t i o n of 20 uCi of H - t h y m i d i n e (15.3 Ci/mole; N e w E n g l a n d N u c l e a r Co., Boston, MA) at 20 h, and t h e i r livers w e r e e x c i s e d at 22 h under a n a e s t h e s i a with e t h e r for d e t e r m i n a t i o n of h e p a t i c DNA synthesis.
~
D e t e r m i n a t i o n s of h e p a t i c a c t i v i t i e s of and p r o t e i n c o n t e n t r and DNA s y n t h e s i s
ODC
and
SAT t p u t r e s c i n e
These d e t e r m i n a t i o n s w e r e d o n e as p r e v i o u s l y d e s c r i b e d (5). The e x c i s e d liver was h o m o g e n i z e d w i t h 9 v o l u m e s of an i c e - c o l d b u f f e r s o l u t i o n c o n s i s t i n g of 50 mM Tris-HCI, pH 7.5, c o n t a i n i n g 5 mM d i t h i o t h r e i t o l , 40 mM p y r i d o x a l p h o s p h a t e and 4 mM EDTA in an a l l - g l a s s T e n b r o c k t i s s u e grinder. The h o m o g e n a t e was c e n t r i fuged at 100,000 x ~ for 60 min at 4°C and the s u p e r n a t a n t was used for the a s s a y for ODC a c t i v i t y . The a c t i v ~ y was d e t e r m i n e d by m e a s u r i n g the r e l e a s e of I~CO2 from D L - [ 1 - 1 ~ C ] - o r n i t h i n e hydrochloride (61 Ci/mole; A m e r s h a m I n t e r n a t i o n a l plc, A m e r s h a m , UK) (7). To o b t a i n the sample for h e p a t i c SAT a c t i v i t y , the hom o g e n i z a t i o n was p e r f o r m e d w i t h 4 v o l u m e s of i c e - c o l d 0.25 M sucrose s o l u t i o n c o n t a i n i n g 50 mM Tris-HCl, pH 7.5, 25 mM KCI and 5 mM MgCI2, the h o m o g e n a t e was c e n t r i f u g e d at 100,000 x ~ for 60 min at 4°C, and the s u p e r n a t a n t was collected. The a c t i v i t y was d e t e r m i n e d w i t h the s u ~ % r n a t a n t by m e a s u r i n g the i n c o r p o r a t i o n of r a d i o a c t i v i t y from [ 1 - ' ~ C ] - a c e t y l - C o A (48.1 Ci/mole; New E n g l a n d Nuclear) into a c e t y l s p e r m i d i n e d u r i n g a 10 min i n c u b a t i o n at 30°C (8). H e p a t i c p u t r e s c i n e c o n t e n t was d e t e r m i n e d by O s h i m a ' s m e t h o d
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Hormones on Liver Putrescine Production
79
(9). The s u p e r n a t a n t o b t a i n e d a f t e r h o m o g e n i z a t i o n w i t h 4 v o l u m e s of 0.5 N p e r c h l o r i c a c i d and c e n t r i f u g a t i o n at 1 0 , 0 0 0 x H for 20 min at 4°C, was a p p l i e d on a c a t i o n e x c h a n g e r e s i n C K - 1 0 U (Mitsubishi Chemical, Tokyo, Japan) column, and was eluted with a g r a d i e n t of 0 . 9 - 2 . 5 M s o d i u m c h l o r i d e in 0.28 M a c e t i c acid buffer, pH 4.9, c o n t a i n i n g 5% a c e t o n i t r i l e at 60°C. The p o l y a m i n e s were quantitated by m e a n s of t h e o - p h t h a l a l d e h y d e reaction. H e p a t i c p r o t e i n c o n t e n t was m e a s u r e d b y B r a d f o r d ' s m e t h o d (10). For the d e t e r m i n a t i o n of h e p a t i c DNA s y n t h e s i s , the h o m o g e n i z a t i o n was p e r f o r m e d w i t h 9 v o l u m e s of cold d i s t i l l e d water. F r o m the p e l l e t of the h o m o g e n a t e after t r e a t m e n t w i t h 5% cold t r i c h l o r o a c e t i c acid, DNA was e x t r a c t e d by the m e t h o d of S c h m i d t T h a n h a u s e r - S c h n e i d e r (11), and its r a d i o a c t i v i t y was c o u n t e d in a liquid scintillation c o u n t e r . D N A c o n t e n t of the e x t r a c t w a s m e a s u r e d by B u r t o n ' s m e t h o d 12).
Results E f f e c t s of g l u c a g o n a n d / o r _ i n s u l i n t r e a t m e n t cine c o n t e n t a f t e r p a r t i a l h e p a t e c t o m y
on
hepatic
putres-
W h e n rats w e r e t r e a t e d w i t h c o m b i n e d g l u c a g o n and insulin, h e p a t i c p u t r e s c i n e c o n t e n t was s i g n i f i c a n t l y i n c r e a s e d to 2.2 and 2.5 times that of c o n t r o l rats at 6 and 12 h, r e s p e c t i v e l y . A f t e r t r e a t m e n t w i t h g l u c a g o n alone, the i n c r e a s e was to 1.9 and 1.7 times, r e s p e c t i v e l y ; w h e r e a s w i t h i n s u l i n alone the i n c r e a s e was f o u n d o n l y at 12 h, to 1.8 times (TABLE I).
TABLE
I
E f f e c t s of G l u c a g o n a n d / o r I n s u l i n T r e a t m e n t on H e p a t i c P u t r e s c i n e C o n t e n t a f t e r P a r t i a l H e p a t e c t o m y in Rats
Groups
Putrescine
content
(nmole/g
liver wt)
Hours after h e p a t e c t o m y 6 Control
210
Glucagon
398 ± 85**
439 ± 123
Insulin
255
475
±
55*
661
±
69**%
G l u c a g o n and
Insulin
± 56
12
± 72
456 ± 41##
266
± I06 a
a v a l u e s are the m e a n s ± SD of 4 rats. Mean h e p a t i c p u t r e s cine c o n t e n t (± SD) for 3 n o r m a l rats was 12 ± I n m o l e / g liver wt. *p<0.05, **p<0.01 vs control; ##p<0.01 vs c o n t r o l and insulin; % p < 0 . 0 5 vs g l u c a g o n ; by B o n f f e r o n i ' s test.
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Hormones on Liver Putrescine Production
Vol. 45, No. i, 1989
E f f e c t s of g l u c a g o n a n d / o r i n s u l i n t r e a t m e n t t i e s of O D C a n d SAT a f t e r p a r t i a l h e p a t e c t o m y
on
hepatic
activi-
Hepatic ODC activity after treatment with combined glucagon and i n s u l i n was a b o v e c o n t r o l l e v e l s at 10 and 12 h. T h i s inc r e a s e was s e e n at 12 h a f t e r i n s u l i n t r e a t m e n t ( T A B L E II). H e p a t i c SAT a c t i v i t y was s i g n i f i c a n t l y i n c r e a s e d by t r e a t m e n t w i t h g l u c a g o n a l o n e or c o m b i n e d g l u c a g o n and i n s u l i n at 4 a n d 6 h or 6 h, r e s p e c t i v e l y , but it was not a f f e c t e d by i n s u l i n t r e a t m e n t ( T A B L E III). Effects t e n t at
of p u t r e s c i n e a d m i n i s t r a t i o n on h e p a t i c p u t r e s c i n e c o n 12 h and D N A s y n t h e s i s 22 h a f t e r p a r t i a l h e p a t e c t o m y
W h e n r a t s r e c e i v e d p u t r e s c i n e f r o m 0 to 4 h or f r o m 6 to 10 h, h e p a t i c p u t r e s c i n e c o n t e n t w a s i n c r e a s e d to 1.5 or 2.0 t i m e s t h a t of c o n t r o l r a t s and h e p a t i c D N A s y n t h e s i s , to 1.7 or 1.8 t i m e s t h a t of c o n t r o l rats, r e s p e c t i v e l y ( T A B L E IV).
TABLE
II
E f f e c t s of G l u c a g o n a n d / o r I n s u l i n T r e a t m e n t Ornithine Decarboxylase (ODC) A c t i v i t y a f t e r H e p a t e c t o m y in R a t s
Groups
ODC
activity
Hours 4
(pmole/h/mg
after
6
on H e p a t i c Partial
protein)
hepatectomy 8
10
12
Control
11 42 +195(5)
501 ±114(4)
676 _+228(4)
81 2 ±240(4)
787 a _+201(4)
Glucagon
I 664 ±458(5)
977 ±770(4)
946 _+476(4)
748 ±159(4)
665 _+25(4)
Insulin
11 46 _+210(5)
945 +528(4)
81 8 _+423(4)
1206 ±204(4)
1 345** _+61 (4)
Glucagon and Insulin
1687 ±358(5)
1048 _+485(4)
935 _+152(4)
1366* ±261(4)
I ] 62** _+148(4)
a v a l u e s a r e the m e a n s ± SD. F i g u r e s in p a r e n t h e s e s a r e n u m b e r of rats. M e a n h e p a t i c O D C a c t i v i t y (± SD) for 3 n o r m a l r a t s was 16 ± 10 p m o l e / h / m g p r o t e i n . * p < 0 . 0 5 , * * p < 0 . 0 1 vs c o n t r o l , by B o n f f e r o n i ' s test.
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Hormones on Liver Putrescine Production
TABLE
81
III
E f f e c t s of G ~ u c a g o n a n d / o r I n s u l i n T r e a t m e n t on H e p a t i c S p e r m i d i n e N ' - a c e t y l t r a n s f e r a s e (SAT) A c t i v i t y a f t e r Partial H e p a t e c t o m y in Rats
Groups
SAT a c t i v i t y
Hours
(pmole/10
after
4
min/mg
hepatectomy 6
41±28
Glucagon
91±27"(5)
326±37**(4)
79±34
(4)
Insulin
52±28
(5)
134±24
62±11
(4)
69±28
(5)
231215**(4)
and
insulin
145±21
8
Control
Glucagon
(5)
protein)
(4)
(4)
62±21a(4)
52± 7 (4)
a v a l u e s are the m e a n s ± SD and figures in p a r e n t h e s e s are n u m b e r of rats. M e a n h e p a t i c SAT a c t i v i t y (± SD) for 3 n o r m a l rats was 0.8 ± 0.3 p m o l e / 1 0 m i n / m g protein. *p<0.05, **p<0.01 vs control, by B o n f f e r o n i ' s test.
TABLE
IV
E f f e c t s of P u t r e s c i n e A d m i n i s t r a t i o n on H e p a t i c P u t r e s c i n e C o n t e n t at 12 h and H e p a t i c DNA S y n t h e s i s 22 h a f t e r Partial H e p a t e c t o m y (Hx) in Rats a
Groups
Putrescine content (nmole/g liver wt)
DNA s y n t h e s i s (dpm/ug DNA)
Control
(0-4)
265
±
63
(4)
87.0
Putrescine
(0-4)
396
± 103
(4)
148.0
Control
(6-10)
266
± 100
(4)
89.6
Putrescine
(6-10)
544
± 101"*(4)
158.8
± 34.4
(6)
± 37.8*(5) ± 33.6
(6)
± 53.4*(6)
aIn P u t r e s c i n e (0-4) and P u t r e s c i n e (6-10) or C o n t r o l (0-4) and C o n t r o l (6-10), rats w e r e g i v e n i n t r a p e r i t o n e a l l y I m l / k g body wt of 0.2 m m o l e / m l of p u t r e s c i n e in saline or s a l i n e at 0, 2 and 4 h and at 6, 8 and 10 h a f t e r Hx, resp e c t i v e l y . V a l u e s are the m e a n s ± SD. F i g u r e s in p a r e n t h e ses are n u m b e r of rats. One rat in P u t r e s c i n e (0-4) was k i l l e d by a n e s t h e s i a at the time of Hx. *p<0.05, **p<0.01 vs control, by B o n f f e r o n i ' s test.
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1989
Discussion The p r e s e n t r e s u l t s show that g l u c a g o n or i n s u l i n e n h a n c e d the i n c r e m e n t of p u t r e s c i n e c o n t e n t in r e g e n e r a t i n g rat liver at d i f f e r e n t times a f t e r p a r t i a l h e p a t e c t o m y , and the c o n t e n t was a d d i t i v e l y i n c r e a s e d w h e n the two h o r m o n e s w e r e c o n c o m i t a n t l y dosed. The i n c r e a s e in h e p a t i c a c t i v i t i e s of e n z y m e s r e l a t e d to p u t r e s c i n e p r o d u c t i o n d i f f e r e d d e p e n d i n g on the hormone. P u t r e s cine a d m i n i s t r a t i o n e n h a n c e d h e p a t i c DNA s y n t h e s i s a f t e r p a r t i a l hepatectomy. As p r e v i o u s l y r e p o r t e d (5), h e p a t i c p u t r e s c i n e c o n t e n t depends on h e p a t i c ODC and SAT a c t i v i t i e s a f t e r p a r t i a l h e p a t e c t o my. Thus, the e n h a n c e d i n c r e m e n t of h e p a t i c p u t r e s c i n e c o n t e n t at 6 h a f t e r g l u c a g o n t r e a t m e n t can be a s s u m e d to be the r e s u l t of i n c r e a s e d a c t i v i t i e s of b o t h ODC and SAT, and the e n h a n c e m e n t at 12 h w o u l d be the r e m a i n i n g effect. S i m i l a r l y , the e n h a n c e m e n t by i n s u l i n t r e a t m e n t can be c o n s i d e r e d to r e f l e c t the s t i m u l a t e d h e p a t i c ODC a c t i v i t i e s at 10 and 12 h. A l t h o u g h the p r e c i s e mecha n i s m of s t i m u l a t i o n of the e n z y m e a c t i v i t i e s by e i t h e r h o r m o n e m u s t be f u r t h e r i n v e s t i g a t e d , it is c l e a r that p u t r e s c i n e p r o d u c tion was a d d i t i v e l y s t i m u l a t e d by b o t h h o r m o n e s in r e g e n e r a t i n g liver. P u t r e s c i n e e x o g e n o u s l y d o s e d from 6 to 10 h s t i m u l a t e d hepatic DNA s y n t h e s i s 22 h a f t e r p a r t i a l h e p a t e c t o m y . H e p a t i c p u t r e s cine c o n t e n t at 12 h a f t e r t r e a t m e n t w i t h c o m b i n e d g l u c a g o n and i n s u l i n was c o m p a r a b l e w i t h the c o n t e n t at 12 h a f t e r p u t r e s c i n e a d m i n i s t r a t i o n from 6 to 10 h. H e p a t i c DNA s y n t h e s i s i n c r e a s e d by putrescine administration would a l s o be e x p l i c a b l e on the base of this content. T h e r e f o r e , it is r e a s o n a b l e to c o n s i d e r that the t r e a t m e n t w i t h c o m b i n e d g l u c a g o n and i n s u l i n a f f e c t the DNA synt h e s i s via p u t r e s c i n e p r o d u c t i o n , a l t h o u g h o t h e r p o s s i b i l i t i e s , such as the s t i m u l a t i o n of p r o t e i n s y n t h e s i s (13), c a n n o t be excluded. P u t r e s c i n e a d m i n i s t r a t i o n from 0 to 4 h c a u s e d s i m i l a r results, s u g g e s t i n g that i n c r e a s i n g h e p a t i c p u t r e s c i n e until 12 h m a k e s no d i f f e r e n c e in p r o v o k i n g DNA s y n t h e s i s . This m i g h t be the r e a s o n that g l u c a g o n and insulin, w h i c h s t i m u l a t e p u t r e s c i n e p r o d u c t i o n at d i f f e r e n t times w i t h i n 12 h, show a c o o p e r a t i v e effect on h e p a t i c DNA s y n t h e s i s (I-3). In c o n c l u s i o n , g l u c a g o n and i n s u l i n a d d i t i v e l y e n h a n c e d hepatic p r o d u c t i o n of p u t r e s c i n e a f t e r p a r t i a l h e p a t e c t o m y t h r o u g h s t i m u l a t i o n of e n z y m e a c t i v i t i e s r e l a t e d to its p r o d u c t i o n . This may e x p l a i n the r e a s o n for the c o o p e r a t i v e e f f e c t of two h o r m o n e s on liver r e g e n e r a t i o n . The m e c h a n i s m of s t i m u l a t i o n of the e n z y m e a c t i v i t i e s may d i f f e r b e t w e e n the two h o r m o n e s .
References I. N.L.R. B u c h e r and M.N. S w a f f i e l d , Proc. N a t . A c a d . S c i . U . S . A . 72 1 1 5 7 - 1 1 6 0 (1975). 2. K. T a k a t s u k i , K. F u j i w a r a , S. Hayashi, Y. Ota, M. Torii, S. M i s h i r o , I. Ogata, A. Sakuma, H. Oka and T. Oda, L i f e Sci. 29 2 6 0 9 - 2 6 1 5 (1981).
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Hormones on Liver Putrescine Production
3. J.A. De Diego, L.M. Molina, J. Bujan, P. Simon, M. Gea, A. M e n c h e r o and J.L. B a l i b r e a , Surg. G y n e c o l . O b s t e t . 163 443-447 (1986). 4. C.W. Tabor and H. Tabor, Ann. Rev. Biochem. 53 749-790 (1984). 5. Y. Sato and K. F u j i w a r a , J . B i o c h e m . 104 98-101 (19~8). 6. G.N. H i g g i n s and R.M. A n d e r s o n , Arch. Pathol. 12 186-202 (1931). 7. T. Oka and J.W. Perry, J . B i o l . C h e m . 251, 1 7 3 8 - 1 7 4 4 (1976). 8 P.R. Libby, J.Biol. Chem. 253, 233-237 (1978) 9 T. Oshima, J . B i o l . C h e m . 2 5 4 , 8 7 2 0 - 8 7 2 2 (1979). I0 M.M. B r a d f o r d , A n a l . B i o c h e m . 72 248-254 (1976). II W.C.A. S c h n e i d e r , J . B i o l . C h e m . 254 8 7 2 0 - 8 7 2 2 (1946). 12 K. Burton, Biochem. J. 62 315-323 (1956). 13 K. F u j i w a r a , I. Ogata, Y. Mishiro, Y. Ohta, Y. Oka, K. T a k a t s u k i , Y. Sato, S. H a y a s h i and H. Oka, Scand. J . G a s t r o enterol. 23 567-573 (1988).
83
ADDITIVE
S T I M U L A T I O N OF H E P A T I C P U T R E S C I N E P R O D U C T I O N BY G L U C A G O N AND INSULIN A F T E R P A R T I A L H E P A T E C T O M Y IN RATS Yuzuru
First
Pergamon Press
Sato, Kenji F u j i w a r a * , Itsuro Ogata, T o m o a k i T o m i y a and Hiroshi Oka
D e p a r t m e n t of M e d i c i n e , F a c u l t y of Medicine, U n i v e r s i t y Tokyo, 7-3-I Hongo, B u n k y o - k u , T o k y o 113, J a p a n
of
(Received in final form April 28, 1989) Summary W h e n rats r e c e i v e d g l u c a g o n or insulin every 2 h a f t e r p a r t i a l h e p a t e c t o m y (Hx), h e p a t i c p u t r e s c i n e c o n t e n t was i n c r e a s e d above control levels at 6 and 12 h, r e s p e c t i v e l y . W h e n the two h o r m o n e s were combined, the i n c r e a s e d levels were additive. Hepatic o r n i t h i n e d e c a r b o x y l a s e a c t i v i t y was above c o n t r o l l e v e l s at 12 h a f t e r i n s u l i n t r e a t m e n t . Hepatic spermidine N1-acetyltransferase activity was enh a n c e d at 6 h only w h e n g l u c a g o n was dosed. P u t r e s cine a d m i n i s t r a t i o n from 0 to 4 h or from 6 to 10 h i n c r e a s e d h e p a t i c DNA s y n t h e s i s to similar levels 22 h a f t e r Hx. These r e s u l t s suggest that g l u c a g o n and insulin additively stimulate hepatic putrescine p r o d u c t i o n after Hx. This may e x p l a i n the c o o p e r a tive s t i m u l a t i o n of liver r e g e n e r a t i o n by both hormones. It has been s u g g e s t e d that g l u c a g o n and insulin c o o p e r a t e in s t i m u l a t i n g liver r e g e n e r a t i o n (I-3). The m e c h a n i s m of s t i m u l a tion, however, is still unclear. It is w i d e l y a c c e p t e d that p o l y a m i n e s play an i m p o r t a n t role in cell d i v i s i o n (4). We have o b t a i n e d e v i d e n c e that p u t r e s c i n e is n e c e s s a r y for DNA s y n t h e s i s in liver r e g e n e r a t i o n after partial h e p a t e c t o m y in rats, and is s u p p l i e d not only via the route of c o n v e r s i o n of o r n i t h i n e into p u t r e s c i n e by o r n i t h i n e d e c a r b o x y l a s e (ODC) but also via the c o m p e n s a t o r y route of s p e r m i d i n e conversion into p u t r e s c i n e by spermidine N1-acetyltransferase (SAT) and p o l y a m i n e o x i d a s e (5). G l u c a g o n and insulin, on the o t h e r h a n d , m a y a c t on l i v e r r e g e n e r a t i o n at t h e G 1 - p h a s e of cell c y c l e (2). Thus, both h o r m o n e s m i g h t affect h e p a t i c p u t r e s cine p r o d u c t i o n a f t e r partial h e p a t e c t o m y . In the p r e s e n t paper, we s t u d i e d this p o s s i b i l i t y the m e c h a n i s m of s t i m u l a t i o n by both h o r m o n e s in rats.
*To w h o m
correspondence
should
be addressed.
0024-3205/89 $3.00 +.00 Copyright (c) 1989 Pergamon Press plc
to c l a r i f y
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Materials
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and M e t h o d s
Experiments Male Sprague-Dawley rats (Charles River Japan, Atsugi, Japan) w e i g h i n g 180-200 g w e r e fed a c o m m e r c i a l p e l l e t e d diet and w a t e r ad l i b i t u m in a c a g e at 2 2 ± 2 ° C u n d e r n o r m a l l a b o r a t o r y lighting conditions. B e t w e e n 9:00 and 11:00 am, they u n d e r w e n t t w o - t h i r d s r e s e c t i o n of the liver a c c o r d i n g to the m e t h o d of Higgins and A n d e r s o n (6) u n d e r light ether a n a e s t h e s i a , and w e r e g i v e n w a t e r ad libitum. E x p e r i m e n t I: H e p a t e c t o m i z e d rats were d i v i d e d into 4 groups. In 3 h o r m o n e - t r e a t e d groups, they r e c e i v e d a s u b c u t a n e o u s i n j e c t i o n of 5 m l / k g body wt of 5% g l u c o s e solution c o n t a i n i n g 5 u g / m l of g l u c a g o n (Novo Industri A/S, C o p e n h a gen, Denmark) a n d / o r 8 mU/ml of A c t r a p i d i n s u l i n (Novo) e v e r y 2 h i m m e d i a t e l y a f t e r o p e r a t i o n , until 2 h b e f o r e sacrifice. In one g r o u p (control), they r e c e i v e d a s i m i l a r dose of the g l u c o s e solution alone. At the time of sacrifice, under ether a n a e s t h e s i a , the liver was p e r f u s e d w i t h 12 ml of i c e - c o l d saline. Then the liver was excised, q u i c k l y f r o z e n in liquid nitrogen, and stored at -80°C until it was used for d e t e r m i n a t i o n s of h e p a t i c a c t i v i ties of O D C and SAT and p u t r e s c i n e content. E x p e r i m e n t II: Hep a t e c t o m i z e d rats were d i v i d e d into 4 groups. In 2 groups, they r e c e i v e d an ip i n j e c t i o n of 1 m l / k g body wt of 0.2 m m o l e / m l putrescine dihydrochloride (Wako Pure C h e m i c a l Industries Ltd., Osaka, Japan) s o l u t i o n in saline a d j u s t e d to pH 7.4 by a d d i t i o n of N a O H at 0, 2 and 4 h or at 6, 8 and 10 h after o p e r a t i o n , and in the other 2 groups, they r e c e i v e d the same v o l u m e of saline alone similarly. They were k i l l e d at 12 h for the d e t e r m i n a t i o n of h e p a t i c p u t r e s c i n e content, as above. In a n o t h e r 4 g r o u p s of h e p a t e c t o m i z e d rats f o l l o w e d by t r e a t m e n t with p u t r e s c i n e dihyr o c b l o r i d e or saline, they r e c e i v e d an ip i n j e c t i o n of 20 uCi of H - t h y m i d i n e (15.3 Ci/mole; N e w E n g l a n d N u c l e a r Co., Boston, MA) at 20 h, and t h e i r livers w e r e e x c i s e d at 22 h under a n a e s t h e s i a with e t h e r for d e t e r m i n a t i o n of h e p a t i c DNA synthesis.
~
D e t e r m i n a t i o n s of h e p a t i c a c t i v i t i e s of and p r o t e i n c o n t e n t r and DNA s y n t h e s i s
ODC
and
SAT t p u t r e s c i n e
These d e t e r m i n a t i o n s w e r e d o n e as p r e v i o u s l y d e s c r i b e d (5). The e x c i s e d liver was h o m o g e n i z e d w i t h 9 v o l u m e s of an i c e - c o l d b u f f e r s o l u t i o n c o n s i s t i n g of 50 mM Tris-HCI, pH 7.5, c o n t a i n i n g 5 mM d i t h i o t h r e i t o l , 40 mM p y r i d o x a l p h o s p h a t e and 4 mM EDTA in an a l l - g l a s s T e n b r o c k t i s s u e grinder. The h o m o g e n a t e was c e n t r i fuged at 100,000 x ~ for 60 min at 4°C and the s u p e r n a t a n t was used for the a s s a y for ODC a c t i v i t y . The a c t i v ~ y was d e t e r m i n e d by m e a s u r i n g the r e l e a s e of I~CO2 from D L - [ 1 - 1 ~ C ] - o r n i t h i n e hydrochloride (61 Ci/mole; A m e r s h a m I n t e r n a t i o n a l plc, A m e r s h a m , UK) (7). To o b t a i n the sample for h e p a t i c SAT a c t i v i t y , the hom o g e n i z a t i o n was p e r f o r m e d w i t h 4 v o l u m e s of i c e - c o l d 0.25 M sucrose s o l u t i o n c o n t a i n i n g 50 mM Tris-HCl, pH 7.5, 25 mM KCI and 5 mM MgCI2, the h o m o g e n a t e was c e n t r i f u g e d at 100,000 x ~ for 60 min at 4°C, and the s u p e r n a t a n t was collected. The a c t i v i t y was d e t e r m i n e d w i t h the s u ~ % r n a t a n t by m e a s u r i n g the i n c o r p o r a t i o n of r a d i o a c t i v i t y from [ 1 - ' ~ C ] - a c e t y l - C o A (48.1 Ci/mole; New E n g l a n d Nuclear) into a c e t y l s p e r m i d i n e d u r i n g a 10 min i n c u b a t i o n at 30°C (8). H e p a t i c p u t r e s c i n e c o n t e n t was d e t e r m i n e d by O s h i m a ' s m e t h o d
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Hormones on Liver Putrescine Production
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(9). The s u p e r n a t a n t o b t a i n e d a f t e r h o m o g e n i z a t i o n w i t h 4 v o l u m e s of 0.5 N p e r c h l o r i c a c i d and c e n t r i f u g a t i o n at 1 0 , 0 0 0 x H for 20 min at 4°C, was a p p l i e d on a c a t i o n e x c h a n g e r e s i n C K - 1 0 U (Mitsubishi Chemical, Tokyo, Japan) column, and was eluted with a g r a d i e n t of 0 . 9 - 2 . 5 M s o d i u m c h l o r i d e in 0.28 M a c e t i c acid buffer, pH 4.9, c o n t a i n i n g 5% a c e t o n i t r i l e at 60°C. The p o l y a m i n e s were quantitated by m e a n s of t h e o - p h t h a l a l d e h y d e reaction. H e p a t i c p r o t e i n c o n t e n t was m e a s u r e d b y B r a d f o r d ' s m e t h o d (10). For the d e t e r m i n a t i o n of h e p a t i c DNA s y n t h e s i s , the h o m o g e n i z a t i o n was p e r f o r m e d w i t h 9 v o l u m e s of cold d i s t i l l e d water. F r o m the p e l l e t of the h o m o g e n a t e after t r e a t m e n t w i t h 5% cold t r i c h l o r o a c e t i c acid, DNA was e x t r a c t e d by the m e t h o d of S c h m i d t T h a n h a u s e r - S c h n e i d e r (11), and its r a d i o a c t i v i t y was c o u n t e d in a liquid scintillation c o u n t e r . D N A c o n t e n t of the e x t r a c t w a s m e a s u r e d by B u r t o n ' s m e t h o d 12).
Results E f f e c t s of g l u c a g o n a n d / o r _ i n s u l i n t r e a t m e n t cine c o n t e n t a f t e r p a r t i a l h e p a t e c t o m y
on
hepatic
putres-
W h e n rats w e r e t r e a t e d w i t h c o m b i n e d g l u c a g o n and insulin, h e p a t i c p u t r e s c i n e c o n t e n t was s i g n i f i c a n t l y i n c r e a s e d to 2.2 and 2.5 times that of c o n t r o l rats at 6 and 12 h, r e s p e c t i v e l y . A f t e r t r e a t m e n t w i t h g l u c a g o n alone, the i n c r e a s e was to 1.9 and 1.7 times, r e s p e c t i v e l y ; w h e r e a s w i t h i n s u l i n alone the i n c r e a s e was f o u n d o n l y at 12 h, to 1.8 times (TABLE I).
TABLE
I
E f f e c t s of G l u c a g o n a n d / o r I n s u l i n T r e a t m e n t on H e p a t i c P u t r e s c i n e C o n t e n t a f t e r P a r t i a l H e p a t e c t o m y in Rats
Groups
Putrescine
content
(nmole/g
liver wt)
Hours after h e p a t e c t o m y 6 Control
210
Glucagon
398 ± 85**
439 ± 123
Insulin
255
475
±
55*
661
±
69**%
G l u c a g o n and
Insulin
± 56
12
± 72
456 ± 41##
266
± I06 a
a v a l u e s are the m e a n s ± SD of 4 rats. Mean h e p a t i c p u t r e s cine c o n t e n t (± SD) for 3 n o r m a l rats was 12 ± I n m o l e / g liver wt. *p<0.05, **p<0.01 vs control; ##p<0.01 vs c o n t r o l and insulin; % p < 0 . 0 5 vs g l u c a g o n ; by B o n f f e r o n i ' s test.
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E f f e c t s of g l u c a g o n a n d / o r i n s u l i n t r e a t m e n t t i e s of O D C a n d SAT a f t e r p a r t i a l h e p a t e c t o m y
on
hepatic
activi-
Hepatic ODC activity after treatment with combined glucagon and i n s u l i n was a b o v e c o n t r o l l e v e l s at 10 and 12 h. T h i s inc r e a s e was s e e n at 12 h a f t e r i n s u l i n t r e a t m e n t ( T A B L E II). H e p a t i c SAT a c t i v i t y was s i g n i f i c a n t l y i n c r e a s e d by t r e a t m e n t w i t h g l u c a g o n a l o n e or c o m b i n e d g l u c a g o n and i n s u l i n at 4 a n d 6 h or 6 h, r e s p e c t i v e l y , but it was not a f f e c t e d by i n s u l i n t r e a t m e n t ( T A B L E III). Effects t e n t at
of p u t r e s c i n e a d m i n i s t r a t i o n on h e p a t i c p u t r e s c i n e c o n 12 h and D N A s y n t h e s i s 22 h a f t e r p a r t i a l h e p a t e c t o m y
W h e n r a t s r e c e i v e d p u t r e s c i n e f r o m 0 to 4 h or f r o m 6 to 10 h, h e p a t i c p u t r e s c i n e c o n t e n t w a s i n c r e a s e d to 1.5 or 2.0 t i m e s t h a t of c o n t r o l r a t s and h e p a t i c D N A s y n t h e s i s , to 1.7 or 1.8 t i m e s t h a t of c o n t r o l rats, r e s p e c t i v e l y ( T A B L E IV).
TABLE
II
E f f e c t s of G l u c a g o n a n d / o r I n s u l i n T r e a t m e n t Ornithine Decarboxylase (ODC) A c t i v i t y a f t e r H e p a t e c t o m y in R a t s
Groups
ODC
activity
Hours 4
(pmole/h/mg
after
6
on H e p a t i c Partial
protein)
hepatectomy 8
10
12
Control
11 42 +195(5)
501 ±114(4)
676 _+228(4)
81 2 ±240(4)
787 a _+201(4)
Glucagon
I 664 ±458(5)
977 ±770(4)
946 _+476(4)
748 ±159(4)
665 _+25(4)
Insulin
11 46 _+210(5)
945 +528(4)
81 8 _+423(4)
1206 ±204(4)
1 345** _+61 (4)
Glucagon and Insulin
1687 ±358(5)
1048 _+485(4)
935 _+152(4)
1366* ±261(4)
I ] 62** _+148(4)
a v a l u e s a r e the m e a n s ± SD. F i g u r e s in p a r e n t h e s e s a r e n u m b e r of rats. M e a n h e p a t i c O D C a c t i v i t y (± SD) for 3 n o r m a l r a t s was 16 ± 10 p m o l e / h / m g p r o t e i n . * p < 0 . 0 5 , * * p < 0 . 0 1 vs c o n t r o l , by B o n f f e r o n i ' s test.
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Hormones on Liver Putrescine Production
TABLE
81
III
E f f e c t s of G ~ u c a g o n a n d / o r I n s u l i n T r e a t m e n t on H e p a t i c S p e r m i d i n e N ' - a c e t y l t r a n s f e r a s e (SAT) A c t i v i t y a f t e r Partial H e p a t e c t o m y in Rats
Groups
SAT a c t i v i t y
Hours
(pmole/10
after
4
min/mg
hepatectomy 6
41±28
Glucagon
91±27"(5)
326±37**(4)
79±34
(4)
Insulin
52±28
(5)
134±24
62±11
(4)
69±28
(5)
231215**(4)
and
insulin
145±21
8
Control
Glucagon
(5)
protein)
(4)
(4)
62±21a(4)
52± 7 (4)
a v a l u e s are the m e a n s ± SD and figures in p a r e n t h e s e s are n u m b e r of rats. M e a n h e p a t i c SAT a c t i v i t y (± SD) for 3 n o r m a l rats was 0.8 ± 0.3 p m o l e / 1 0 m i n / m g protein. *p<0.05, **p<0.01 vs control, by B o n f f e r o n i ' s test.
TABLE
IV
E f f e c t s of P u t r e s c i n e A d m i n i s t r a t i o n on H e p a t i c P u t r e s c i n e C o n t e n t at 12 h and H e p a t i c DNA S y n t h e s i s 22 h a f t e r Partial H e p a t e c t o m y (Hx) in Rats a
Groups
Putrescine content (nmole/g liver wt)
DNA s y n t h e s i s (dpm/ug DNA)
Control
(0-4)
265
±
63
(4)
87.0
Putrescine
(0-4)
396
± 103
(4)
148.0
Control
(6-10)
266
± 100
(4)
89.6
Putrescine
(6-10)
544
± 101"*(4)
158.8
± 34.4
(6)
± 37.8*(5) ± 33.6
(6)
± 53.4*(6)
aIn P u t r e s c i n e (0-4) and P u t r e s c i n e (6-10) or C o n t r o l (0-4) and C o n t r o l (6-10), rats w e r e g i v e n i n t r a p e r i t o n e a l l y I m l / k g body wt of 0.2 m m o l e / m l of p u t r e s c i n e in saline or s a l i n e at 0, 2 and 4 h and at 6, 8 and 10 h a f t e r Hx, resp e c t i v e l y . V a l u e s are the m e a n s ± SD. F i g u r e s in p a r e n t h e ses are n u m b e r of rats. One rat in P u t r e s c i n e (0-4) was k i l l e d by a n e s t h e s i a at the time of Hx. *p<0.05, **p<0.01 vs control, by B o n f f e r o n i ' s test.
82
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Vol. 45, No. I,
1989
Discussion The p r e s e n t r e s u l t s show that g l u c a g o n or i n s u l i n e n h a n c e d the i n c r e m e n t of p u t r e s c i n e c o n t e n t in r e g e n e r a t i n g rat liver at d i f f e r e n t times a f t e r p a r t i a l h e p a t e c t o m y , and the c o n t e n t was a d d i t i v e l y i n c r e a s e d w h e n the two h o r m o n e s w e r e c o n c o m i t a n t l y dosed. The i n c r e a s e in h e p a t i c a c t i v i t i e s of e n z y m e s r e l a t e d to p u t r e s c i n e p r o d u c t i o n d i f f e r e d d e p e n d i n g on the hormone. P u t r e s cine a d m i n i s t r a t i o n e n h a n c e d h e p a t i c DNA s y n t h e s i s a f t e r p a r t i a l hepatectomy. As p r e v i o u s l y r e p o r t e d (5), h e p a t i c p u t r e s c i n e c o n t e n t depends on h e p a t i c ODC and SAT a c t i v i t i e s a f t e r p a r t i a l h e p a t e c t o my. Thus, the e n h a n c e d i n c r e m e n t of h e p a t i c p u t r e s c i n e c o n t e n t at 6 h a f t e r g l u c a g o n t r e a t m e n t can be a s s u m e d to be the r e s u l t of i n c r e a s e d a c t i v i t i e s of b o t h ODC and SAT, and the e n h a n c e m e n t at 12 h w o u l d be the r e m a i n i n g effect. S i m i l a r l y , the e n h a n c e m e n t by i n s u l i n t r e a t m e n t can be c o n s i d e r e d to r e f l e c t the s t i m u l a t e d h e p a t i c ODC a c t i v i t i e s at 10 and 12 h. A l t h o u g h the p r e c i s e mecha n i s m of s t i m u l a t i o n of the e n z y m e a c t i v i t i e s by e i t h e r h o r m o n e m u s t be f u r t h e r i n v e s t i g a t e d , it is c l e a r that p u t r e s c i n e p r o d u c tion was a d d i t i v e l y s t i m u l a t e d by b o t h h o r m o n e s in r e g e n e r a t i n g liver. P u t r e s c i n e e x o g e n o u s l y d o s e d from 6 to 10 h s t i m u l a t e d hepatic DNA s y n t h e s i s 22 h a f t e r p a r t i a l h e p a t e c t o m y . H e p a t i c p u t r e s cine c o n t e n t at 12 h a f t e r t r e a t m e n t w i t h c o m b i n e d g l u c a g o n and i n s u l i n was c o m p a r a b l e w i t h the c o n t e n t at 12 h a f t e r p u t r e s c i n e a d m i n i s t r a t i o n from 6 to 10 h. H e p a t i c DNA s y n t h e s i s i n c r e a s e d by putrescine administration would a l s o be e x p l i c a b l e on the base of this content. T h e r e f o r e , it is r e a s o n a b l e to c o n s i d e r that the t r e a t m e n t w i t h c o m b i n e d g l u c a g o n and i n s u l i n a f f e c t the DNA synt h e s i s via p u t r e s c i n e p r o d u c t i o n , a l t h o u g h o t h e r p o s s i b i l i t i e s , such as the s t i m u l a t i o n of p r o t e i n s y n t h e s i s (13), c a n n o t be excluded. P u t r e s c i n e a d m i n i s t r a t i o n from 0 to 4 h c a u s e d s i m i l a r results, s u g g e s t i n g that i n c r e a s i n g h e p a t i c p u t r e s c i n e until 12 h m a k e s no d i f f e r e n c e in p r o v o k i n g DNA s y n t h e s i s . This m i g h t be the r e a s o n that g l u c a g o n and insulin, w h i c h s t i m u l a t e p u t r e s c i n e p r o d u c t i o n at d i f f e r e n t times w i t h i n 12 h, show a c o o p e r a t i v e effect on h e p a t i c DNA s y n t h e s i s (I-3). In c o n c l u s i o n , g l u c a g o n and i n s u l i n a d d i t i v e l y e n h a n c e d hepatic p r o d u c t i o n of p u t r e s c i n e a f t e r p a r t i a l h e p a t e c t o m y t h r o u g h s t i m u l a t i o n of e n z y m e a c t i v i t i e s r e l a t e d to its p r o d u c t i o n . This may e x p l a i n the r e a s o n for the c o o p e r a t i v e e f f e c t of two h o r m o n e s on liver r e g e n e r a t i o n . The m e c h a n i s m of s t i m u l a t i o n of the e n z y m e a c t i v i t i e s may d i f f e r b e t w e e n the two h o r m o n e s .
References I. N.L.R. B u c h e r and M.N. S w a f f i e l d , Proc. N a t . A c a d . S c i . U . S . A . 72 1 1 5 7 - 1 1 6 0 (1975). 2. K. T a k a t s u k i , K. F u j i w a r a , S. Hayashi, Y. Ota, M. Torii, S. M i s h i r o , I. Ogata, A. Sakuma, H. Oka and T. Oda, L i f e Sci. 29 2 6 0 9 - 2 6 1 5 (1981).
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Hormones on Liver Putrescine Production
3. J.A. De Diego, L.M. Molina, J. Bujan, P. Simon, M. Gea, A. M e n c h e r o and J.L. B a l i b r e a , Surg. G y n e c o l . O b s t e t . 163 443-447 (1986). 4. C.W. Tabor and H. Tabor, Ann. Rev. Biochem. 53 749-790 (1984). 5. Y. Sato and K. F u j i w a r a , J . B i o c h e m . 104 98-101 (19~8). 6. G.N. H i g g i n s and R.M. A n d e r s o n , Arch. Pathol. 12 186-202 (1931). 7. T. Oka and J.W. Perry, J . B i o l . C h e m . 251, 1 7 3 8 - 1 7 4 4 (1976). 8 P.R. Libby, J.Biol. Chem. 253, 233-237 (1978) 9 T. Oshima, J . B i o l . C h e m . 2 5 4 , 8 7 2 0 - 8 7 2 2 (1979). I0 M.M. B r a d f o r d , A n a l . B i o c h e m . 72 248-254 (1976). II W.C.A. S c h n e i d e r , J . B i o l . C h e m . 254 8 7 2 0 - 8 7 2 2 (1946). 12 K. Burton, Biochem. J. 62 315-323 (1956). 13 K. F u j i w a r a , I. Ogata, Y. Mishiro, Y. Ohta, Y. Oka, K. T a k a t s u k i , Y. Sato, S. H a y a s h i and H. Oka, Scand. J . G a s t r o enterol. 23 567-573 (1988).
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