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Effect of dibutyryl cyclic AMP on glucose-6-phosphatase activity in human fetal liver explants
500
Biochimica et Biophysica Acta, 343 ( 1 9 7 4 ) 5 0 0 - - 5 0 9 © Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s
BBA 2 7 4 0 0
EFFECT OF DIBUTYRYL CYCLIC AMP ON GLUCOSE-6-PHOSPHATASE ACTIVITY IN HUMAN FETAL LIVER EXPLANTS
A L A N L. S C H W A R T Z a'*, N I E L S C.R. R~,IH:~ a a n d T H E O D O R E W. R A L L b
aDepartment of Medical Chemistry, University of Helsinki, Helsinki (Finland) and bDepartment of Pharmacology, Case Western Reserve University, Cleveland, Ohio (U.S.A.) (Received D e c e m b e r 7 t h , 1 9 7 3 )
Summary Glucose-6-phosphatase (EC 3.1.3.9) activity in human fetal liver remains constant at 8--28 nmoles/min per mg protein from the 8th week of gestation to at least week 28 and this value is approximately 25--35% of that found in the adult. This enzyme activity was well maintained for 2--3 days in organ culture of fetal liver explants. Incubation with dibutyryl cyclic AMP (0.1 mM) and theophylline (0.5 mM) increased glucose-6-phosphatase activity 4--8-fold within 24 h. Theophylline alone was ineffective, but markedly potentiated the effects of dibutyryl cyclic AMP. This increase in enzyme activity was completely abolished by simultaneous incubation with cycloheximide or actinomycin D. Insulin clearly decreased glucose-6-phosphatase activity in control tissues after 24 h incubation and tended to diminish the elevated glucose-6-phosphatase activity which resulted from pre-incubation with dibutyryl cyclic AMP. The smallest specimen obtained (36 mm crown-rump length = 6 weeks gestation) wa s capable of elevating glucose-6-phosphatase activity more than 3-fold in response to dibutyryl cyclic AMP incubation, suggesting that the human fetal liver has the competence to respond to hormonal agents at a very early stage of development.
Introduction
Since the initial studies demonstrated that adult rat liver glucose-6-phosphatase (EC 3.1.3.9) activity could be increased by steroid administration, fasting/starvation or diabetes (Langdon and Weakley [1]; Weber et al. [2] ), many investigations have examined the ability of various hormones to regulate
* Present address: D e p a r t m e n t of P h a r m a c o l o g y , Case Western Reserve University, Cleveland, Ohio, U.S.A.
501 this activity. Administration of insulin to adult rats was not only capable of decreasing the elevated glucose-6-phosphatase activity associated with diabetes, but was also capable of reducing the glucose-6-phosphatase activity in normal rats in vivo (Ashmore et al. [3], Fisher and Stetten [4] ). Glucagon, however, was ineffective in altering hepatic glucose-6-phosphatase activity in the dog in vivo (Cahill et al. [5] ). Recently much attention has focused on the mechanisms involved in the regulation of this activity by steroid hormones and insulin (see Weber and Cantero [6] ). The development of glucose-6-phosphatase activity in mammalian liver has been documented for a wide variety of species including the rat (Weber and Cantero [6], Dawkins [7] ), mouse (Dawkins [8] ), pig (Mersmann [9] ), guinea pig (Nemeth [10], Lea and Walker [11] ), rabbit (Dawkins [8] ), and lamb (Dawkins [7], Ballard and Oliver [12] ). In all of these species the hepatic glucose-6-phosphatase activity is undetectable until just prior to birth, followed by a dramatic increase in activity in the initial postnatal period surpassing that level found in the adult animal (Dawkins [8], Lea and Walker [11] ). This postnatal increase has been attributed to synthesis of protein de novo (Dawkins [13] ). In addition, Greengard and her associates have suggested that glucagon and cyclic AMP are the primary physiological trigger mechanism since administration of these agents to fetuses in utero causes a premature increase in glucose-6-phosphatase activity (Greengard and Dewey [14], Greengard [15]). Dawkins [13] was able to demonstrate that insulin administration to the newborn rat retarded the normal postnatal increase in activity, although insulin administration to fetuses in utero did not alter the glucose-6-phosphatase activity (Greengard [ 15 ] ). In contrast to those species described above, the human fetal liver contains detectable glucose-6-phosphatase activity by mid-gestation (Villee [16], Aurrichio and Rigillo [17], Gennser et al. [18] ) and at this stage of development is already 25--50% of the level in adult liver. The only other species to exhibit significant glucose-6-phosphatase activity in the fetal liver is the rhesus monkey (Dawkins [8], ). No studies have reported the regulation of this activity in either of these species. The ability of the organ culture system to maintain fetal liver explants in a chemically defined system devoid of circulating factors present in utero for a period of days allows one to examine the control of glucose-6-phosphatase activity by hormonal agents. The present study describes the regulation of glucose-6-phosphatase activity in human fetal liver explants by cyclic AMP. Materials and Methods Materials
All chemicals were of analytical grade and were purchased from Sigma Chemical Co., St. Louis, Mo., U.S.A. except: cycloheximide from Nutritional Biochemicals, Cleveland, Ohio, U.S.A.; actinomycin D from Merck, Sharp and Dohme, Rahway, N.J., U.S.A.; dibutyryl cyclic AMP from Calbiochem, Los Angeles, Calif., U.S.A.; insulin (porcine m o n o c o m p o n e n t lot number MC-S821506) from Novo Industri, Copenhagen, Denmark. The culture medium was a modified Eagle's minimal essential medium in Hank's balanced salt solution (Schwartz [19] ) and was prepared by Orion Oy/Ab, Helsinki, Finland.
502
Tissues and organ culture Tissue was obtained at legal therapeutic abortion by hysterotomy*. A portion of the tissue was rapidly excised and placed into ice-cold culture medium. The remainder was quick frozen in liquid N2. A complete description of the culture system has been published previously (R~iih~i et al. [ 2 0 ] , Schwartz [19] ). Briefly, explants less than 1 mm 3 were placed at the media-gas (02/CO2 ; 95:5, v/v) interface for up to 4 days in culture. All tissues were preincubated for 20--24 h before the experiments began. Additions to the culture medium were made at appropriate times in volumes of less than 0.125 ml (less than 1% of the total volume), so as not to alter the composition of the medium. Furthermore, most agents were prepared in culture medium before addition. At the appropriate times in culture the explants were removed and quick frozen over liquid N2. All samples were stored at --80 ° C until assayed for glucose-6-phosphatase activity. Since the availability of tissue dictated the range of sample sizes examined and since no t w o specimens were obtained at the same time, each series of culture dishes prepared from one tissue had to constitute a complete experiment. Thus, many factors, including the various tissue ages as well as unaccountable variations occurring during pregnancy and surgery, make if difficult to compare absolute values from one culture tissue to another (e.g. dose response curves from t w o tissues demonstrate the same pattern, although the absolute values are different). Glucose-6-phosphatase activity Glucose-6-phosphatase activity was determined as described earlier (Schwartz [19] ) in which a piece of quick-frozen liver or a sample of 25 pooled explants was homogenized in glass-distilled water and a portion of the whole homogenate was incubated in a system containing 40 mM glucose 6-phosphate, 7 mM histidine, 1 mM EDTA, pH 6.5, for 20 min at 37°C. The release of Pi was measured according to Fiske and S u b b a R o w [21]. This procedure was linear as a function of time and amount of homogenate assayed. The homogenate protein concentration was determined according to Lowry et al. [22] as modified by Hartree [23] with bovine serum albumin as a standard. The glucose-6-phosphatase activity is expressed as nmoles Pi released/min per mg protein. Statistical significance is expressed as P values from Student's t-test; values of P > 0.10 are considered n o t significant. Results Glucose-6-phosphatase activity is present in human fetal liver in the smallest specimen examined (60 mm crown-rump length = 8 weeks gestation**) and remains at a b o u t the same level throughout the first two-thirds of gestation (Fig. 1). Earlier reports of glucose-6-phosphatase activity in human fetal liver were confined to few specimens (Villee [16] ) or those greater than 20 weeks
* **
N o n e o f t h e i n v e s t i g a t o r s p a r t i c i p a t e d in t h e d e c i s i o n t o i n t e r r u p t p r e g n a n c y . G e s t a t i o n a l a g e w a s d e t e r m i n e d f ~ o m t h e c r o w n - r u m p l e n g t h n o m o g r a m o f T a n i m u r a et a t . [ 2 4 ] .
503 28" 24-
==-
~
~o-
~. 16o. ~
12-
i ,~
B-
~- ~
~.
"4
~
0 Crown-rump
Length (cm)
Fig. 1. N o r m a l d e v e l o p m e n t o f g l u c o s e - 6 - p h o s p h a t a s e a c t i v i t y in h u m a n fetal liver. Livers w e r e excised f r o m f e t u s e s d e l i v e r e d b y h y s t e r o t o m y a n d w e r e r a p i d l y f r o z e n in liquid N 2. E n z y m e a c t i v i t y w a s l a t e r d e t e r m i n e d f o r e a c h s p e c i m e n . E a c h p o i n t r e p r e s e n t s o n e liver. G e s t a t i o n a l age v a r i e d f r o m 8 w e e k s ( 6 0 m m c r o w n - r u m p l e n g t h ) t o 28 w e e k s .
gestation (Aurrichio and Rigillo [17] ). Recently, Gennser et al. [18] determined glucose-6-phosphatase activity in tissues of 13--23 weeks gestation. The results described in Fig. 1 are consistent with these previous reports, both in terms of relative activities during fetal development and absolute activity values. When human fetal liver explants were placed in organ culture only a small decrease in activity was observed over the initial 48 h in culture (Fig. 2). The addition of dibutyryl cyclic AMP and theophylline at concentrations which 100 '
8O
"I.o j _~
"..,!
40
o
g
=o.
0 0 Hours
in C u l t u r e
Fig. 2. T i m e c o u r s e o f d i b u t y r y l c y c l i c AMP plus t h e o p h y l l i n e o n g l u c o s e - f i - p h o s p h a t a s e a c t i v i t y in h u m a n f e t a l liver e x p l a n t s . E x p l a n t s w e r e p r e p a r e d f r o m a s p e c i m e n o f 1 7 0 m m c r o w n - r u m p l e n g t h . A f t e r 24 h p r e i n c u b a t i o n , d i b u t y r y l cyclic AMP a n d t h e o p h y l i i n e w e r e a d d e d t o s o m e c u l t u r e dishes ( e - - e ) to y i e l d final c o n c e n t r a t i o n s o f 0.1 m M a n d 0 . 5 m M , r e s p e c t i v e l y . No a d d i t i o n w a s m a d e t o t h e c o n t r o l dishes ( o - - o ) . A t t h e a p p r o p r i a t e t i m e s , t h e e x p l a n t s w e r e r e m o v e d a n d q u i c k f r o z e n o v e r l i q u i d N 2. E a c h d e t e r m i n a t i o n w a s p e r f o r m e d o n 2 5 p o o l e d e x p l a n t s . E a c h figure r e p r e s e n t s t h e m e a n + S.E. ( f o u r d e t e r m i n a t i o n s ) . T h e d i b u t y r y l cyclic A M P - c o n t a i n i n g s a m p l e s are s i g n i f i c a n t l y g r e a t e r t h a n t h e c o n t r o l s at all t i m e p e r i o d s e x a m i n e d (P < 0 . 0 1 ) .
504 TABLE I E F F E C T OF T H E O P H Y L L I N E ON D I B U T Y R Y L C Y C L I C A M P - S T I M U L A T E D G L U C O S E - 6 - P H O S P H A T A S E A C T I V I T Y IN H U M A N F E T A L L I V E R E X P L A N T S All tissues w e r e p r e i n c u b a t e d for 24 h, a f t e r w h i c h d i b u t y r y l cyclic AMP a n d / o r t h e o p h y l i i n e was a d d e d . 24 h l a t e r all tissues w e r e r e m o v e d f r o m c u l t u r e . 25 p o o l e d e x p l a n t s w e r e r e q u i r e d f o r e a c h analysis. E a c h figure r e p r e s e n t s t h e m e a n +_ S.E. ( n u m b e r of d e t e r m i n a t i o n s in p a r e n t h e s e s ) . T h e c r o w n - r u m p l e n g t h of this s p e c i m e n w a s 54 r a m . N.S., n o t significant. Agent
Final concentration
T h e ophylline (0.5 m M )
Glucose-6-phosphatase activity ( n m o l e s / m i n p e r mg protein)
None None D i b u t y r y l cyclic AMP D i b u t y r y l cyclic AMP D i b u t y r y l cyclic AMP
--0.1 m M 0.1 m M 0.01 m M
+ + +
17.91 19.54 54.55 70.89 33.94
_+ 4 . 5 5 ± 1.91 + 7.09 -+ 2.25 _+ 5.88
(4) (4) (4) (4) (2)
P
}
n.s.
}
0.03
} ~0.01
were maximal for effects on glycogen metabolism and amino acid uptake (Schwartz [25] ) (0.1 mM and 0.5 mM, respectively) increased glucose-6-phosphatase activity clearly by 8 h after addition (Fig. 2). By 24 h after addition, the activity reached in this experiment was more than 5-fold the control value. Under these same conditions, all livers examined responded in a similar manner with glucose-6-phosphatase activity increases ranging from 3.6-fold (Table I) to 7.7-fold (Table III). In the one experiment in which the dibutyryl cyclic AMP was present for more than 24 h, the glucose-6-phosphatase activity continued to increase at the 48th hour, although this value was not statistically greater than 50-
-
40-
~
.o
3o-. •
¢
20.
o 10-
0
6
.,t o.~o~
o.b~
o'.~ Dibutyryl
Cyclic
~:o AMP
(raM)
Fig. 3. E f f e c t o f d i b u t y r y l cyclic AMP in t h e p r e s e n c e of t h e o p h y l l i n e o n g l u c o s e - 6 - p h o s p h a t a s e a c t i v i t y in h u m a n f e t a l liver e x p l a n t s . E x p l a n t s w e r e P r e p a r e d f r o m a s p e c i m e n of 1 6 5 m m c r o w n - r u m p l e n g t h a n d i n c u b a t e d 24 h w i t h t h e d i h u t y r y l cyclic A M P a f t e r t h e s t a n d a r d p r e i n c u b a t i o n . F o r o t h e r details see t h e l e g e n d t o Fig. 2. E a c h figure r e p r e s e n t s t h e m e a n -+ S.E. ( f o u r d e t e r m i n a t i o n s ) . All d i b u t y r y l cyclic A M P - c o n t a i n i n g s a m p l e s are significantly g r e a t e r t h a n t h e c o n t r o l (P ~ 0 . 0 1 ) . T h e c o n t r o l a n d experim e n t a l dishes c o n t a i n e d 0.5 m M t h e o p h y l l i n e .
505 that after 24 h (P ~ 0.09). These observations demonstrate the marked effect of cyclic AMP as a stimulator of glucose-6-phosphatase activity in human fetal liver. Dose relationships for the cyclic nucleotide were examined and yielded the results depicted in Fig. 3. 0.1 mM dibutyryl cyclic AMP in the presence of theophylline was found to be the maximally effective concentration, with smaller activity increases evident after incubation with either lesser (0.01 mM) or greater (1.0 mM) concentrations of the cyclic nucleotide (Fig. 3). Theophylline alone did not produce marked increases in the glucose-6-phosphatase activity, but potentiated the effects of 0.1 mM dibutyryl cyclic AMP (Table I). This effect is similar to that observed for theophylline effects in human fetal liver explants in examinations of amino acid uptake (Schwartz [25] ) and enzyme induction (R~iihti and Schwartz [26] ). Some insight into the mechanism of the cyclic AMP effect was gained by examining the effects of actinomycin D and cycloheximide on the dibutyryl cyclic AMP-stimulated increase in glucose-6-phosphatase activity. As seen in Table II, addition of actinomycin D alone to the cultures produced only a slight decrease in enzyme activity, when tissues were removed from the cultures 30 h later. However, the simultaneous addition of the antibiotic with the cyclic nucleotide completely prevented the 5-fold increase which resulted from the dibutyryl cyclic AMP alone (Table II). Cycloheximide was added to the cultures at various times after the addition of dibutyryl cyclic AMP. As seen in Table III, preincubations of 0--4 h before cycloheximide addition did not produce increased glucose-6-phosphatase activity. However, there may be a slight increase in activity in tissues preincubated 4 h when compared to the cycloheximide controls (P = 0.03). The effects of insulin were examined on glucose-6-phosphatase activity in these explants. As seen in Table IV, the addition of insulin at the 40th hour in culture significantly decreased the enzyme activity for tissues examined 24 h later. The effect of insulin on dibutyryl cyclic AMP-stimulated enzyme activity was examined by incubating the tissues with maximal concentrations of the cyclic nucleotide plus theophylline for 24 h, after which the culture medium T A B L E II EFFECT OF ACTINOMYCIN D ON DIBUTYRYL CYCLIC AMP-STIMULATED PHATASE ACTIVITY IN HUMAN FETAL LIVER EXPLANTS
GLUCOSE-6-PHOS-
Tissues were p r e i n c u b a t e d f o r 24 h, after w h i c h d i b u t y r y l cyclic A M P , t h e o p h y l l i n e , o r a c t i n o m y c i n D w e r e a d d e d t o y i e l d f i n a l c o n c e n t r a t i o n s of 0.1 m M , 0 . 5 m M a n d 25 ~ g / m l , respectively. The t i s s u e s w e r e r e m o v e d f r o m c u l t u r e 3 0 h l a t e r . 25 p o o l e d e x p l a n t s w e r e r e q u i r e d for each analysis. Each figure represents the m e a n __. S.E. ( n u m b e r o f d e t e r m i n a t i o n s in parentheses). The c r o w n - r u m p length of this sample w a s 95 mm. Agents
h
Glucose-6-phosphatase activity ( n m o l e s / m i n per mg protein)
None Dibutyryl cyclic AMP + theophylline A c t i n o m y cin D D i b u t y r y l cyclic AMP + theophyHine + a c t i n o m y c i n D
30 30 30 30
16.33+ 2.96 (4) 85.02+ 15.14 (4) 10.23+ 0.81 (3) 8.33 __+ 0.95 (4)
P
I} ~
~0.01 0.06 0.09
506 TABLE III E F F E C T O F C Y C L O H E X I M I D E ON D I B U T Y R Y L C Y C L I C A M P - S T I M U L A T E D G L U C O S E - 6 - P H O S P H A T A S E A C T I V I T Y IN H U M A N F E T A L L I V E R E X P L A N T S All tissues w e r e p r e i n e u b a t e d f o r 24 h b e f o r e e x p o s e d to t h e a g e n t s i n d i c a t e d a n d all tissues w e r e r e m o v e d f r o m c u l t u r e a f t e r e x a c t l y 48 h. T h e final c o n c e n t r a t i o n s w e r e 0.1 m M d i b u t y r y l cyclic AMP; 0.5 m M t h e o p h y l l i n e ; 10 # g / m l c y c l o h e x i m i d e . E a c h figure r e p r e s e n t s t h e m e a n ± S.E. ( f o u r d e t e r m i n a t i o n s ) . P v a l u e s are b y c o m p a r i s o n to t h e 24 h c o n t r o l . T h e s p e c i m e n w a s 1 3 5 m m c r o w n - r u m p l e n g t h . Agent
h
Glucose-6-phosphatase activity (nmoles/min per mg protein)
None None D i b u t y r y l cyclic Cycloheximide D i b u t y r y l cyclic + cyeloheximide D i b u t y r y l cyclic + cycloheximide D i b u t y r y l cyclic + cycloheximide D i b u t y r y l cyclic + cycloheximide D i b u t y r y l cyclic + cycloheximide
0 24 24 24 24 24 24 23.5 24 23 24 22 24 20
17.28 10.48 81.05 7.18
AMP + t h e o p h y l l i n e AMP + t h e o p h y l l i n e AMP + t h e o p h y U i n e AMP + t h e o p h y U i n e AMP
+
theophylline
AMP + t h e o p h y l l i n e
± 0.39 + 1.67 +_ 8.51 ± 1.94
P
~0.01 --
~ 0.01 n.s.
9.35 ± 1.28
n.s.
1 0 . 8 6 + 1.42
n.s.
6.10 ± 1.02
0.03
9 . 6 9 _+ 0.27
n.s.
1 2 . 5 4 _+ 1.39
n.s.
T A B L E IV E F F E C T OF C O M B I N A T I O N S OF I N S U L I N A N D C Y C L O H E X I M I D E W I T H D I B U T Y R Y L C Y C L I C AMP ON T H E G L U C O S E - 6 - P H O S P H A T A S E A C T I V I T Y I N H U M A N F E T A L L I V E R E X P L A N T S E x p l a n t s w e r e p r e p a r e d f r o m a s p e c i m e n of 1 0 5 m m c r o w n - r u m p l e n g t h a n d w e r e p r e i n c u b a t e d f o r 16 h in t h e s t a n d a r d m e d i a . A t this t i m e , e i t h e r d i b u t y r y l cyclic AMP ( 0 . I raM) plus t h e o p h y l l i n e (0.5 m M ) or n o a d d i t i o n s w e r e m a d e to t h e dishes. 2 4 h l a t e r s o m e of t h e dishes h a d t h e m e d i a r e p l a c e d w i t h fresh s t a n d a r d m e d i a ( w i t h o u t a g e n t s ) a n d the tissues rinsed. A g e n t s w e r e t h e n again a d d e d t o s o m e dishes (insulin, 1 u n i t / m l ; c y c l o h e x i m i d e , 10 # g / m l ) . E a c h d e t e r m i n a t i o n r ~ q u i r e d 25 p o o l e d e x p l a n t s . E a c h figure r e p r e s e n t s the m e a n + S.E. ( f o u r d e t e r m i n a t i o n s ) . Glucose-6phosphatase activity (nrnoles/min p e r mE p r o t e i n )
Agent added
Glucose-6phosphatase activity (nmoles/min per mg protein)
16 h 17.56 + 1.48
Media wash
Agent added
40 h None
+
None
-
+
Insulin
-
None None
--
+ +
--
+
-
Dibutyryl
54.35
--
AMP
P
64 h
--
cyclic
Gincose-6phosphatase activity (nmoles/min per mg protein)
+ 2.44
11.36+1.43 ) 6.01 -+ 1 . 4 8 8 6 . 1 1 + 21.2 50-58+7.91 l~
Insulin
3 6 79 ± 3 ° 5 S - <
Cycloheximide
3O.68 + 4 . 2 0
<0.02
°:°I
507
was changed, the tissues rinsed in fresh medium and new additions made to the fresh medium. With no new addition the activity was the same when explants were examined 24 h later, perhaps a result of a gradual increase followed by a gradual decrease in activity. Addition of insulin after the medium change tended to decrease the enzyme activity although it was significant only at the P = 0.08 level. Cycloheximide addition after the medium change similarly produced a decrease in activity, perhaps slightly more marked than the insulin effect ( P < 0.05). Re-addition of dibutyryl cyclic AMP plus theophylline further markedly increased the glucose-6-phosphatase activity during the final 24 h of incubation (Table IV). The development of the liver's competence to respond to cyclic AMP by increasing the glucose-6-phosphatase activity was examined in early human fetal livers. The youngest specimen examined was 36 mm crown-rump length (6 weeks gestational age). 24 h incubation with dibutyryl cyclic AMP (0.1 mM) and theophylline (0.5 mM) elevated the control (17.38 + 3.64 (4) nmoles/min per mg protein) activity to 55.32 + 6.69 (4) nmoles/min per mg protein. Younger samples are virtually unobtainable and contain but a few milligrams of tissue. Thus by the 6th gestational week, the fetal liver has the ability to respond to hormonal agents. This same pattern of response has been observed with regard to the ability of the human fetal liver early in gestation (6 weeks) to respond to cyclic AMP in terms of amino acid uptake (Schwartz [25] ) and glycogenolysis (Schwartz, A.L., R~iihR, N.C.R. and Rall, T.W., unpublished) and has been adequately discussed therein*. Discussion
Many of the metabolic parameters involved in the regulation of glucose output by the mammalian liver have been examined in relation to their control by glucagon and cyclic AMP on one hand and insulin on the other. In adult rat liver, glycogenolysis is promoted by glucagon/cyclic AMP (Villar-Pilasi et al. [27] ), whereas insulin stimulates glycogen formation (Exton and Park [28] ). Glucagon/cyclic AMP stimulate gluconeogenesis from [14 C]lactat e or [~ 4C]alanine (Mallette et al. [29] ), while insulin diminishes this effect (Exton and Park [28] ). In addition, urea formation (Exton and Park [28] ) appears to be controlled in opposite directions by these agents. Induction of hepatic enzymes** represents another phenomenon associated with opposite control by
*
**
A specimen ( c r o w n - r u m p length = 65 m m ) was obtained f r o m a w o m a n who had be e n receiving 5 m g o f a s y n t h e t i c p r o g e s t e r o n e / d a y f o r t h e 6 d a y s p r e v i o u s t o a b o r t i o n . The c o n t r o l v a l u e s f o r t h e f e t a l Hver g l u c o s e - 6 - p h o s p h a t a s e a c t i v i t y w e r e w i t h i n t h e n o r m a l r a n g e , as w e r e t h e v a l u e s in c o n t r o l c u l t u r e s ( 2 3 . 2 3 ± 6.41 (4) n m o l e s / m i n p e r m g p r o t e i n ) . H o w e v e r , t h e r e s p o n s e t o 24 h i n c u b a t i o n w i t h d i b u t y r y l cyclic AMP (0.1 m M ) a n d t h e o p h y l i i n e ( 0 . 5 m M ) r e s u l t e d in t h e largest activity observed in a n y tissue u n d e r the s a m e c o n d i t i o n s o f cultuxe ( 1 1 7 . 2 5 + 1 4 . 9 (4) n m o l e s / m i n per mg protein). A second specimen ( c r o w n - r u m p length = 150 ram) was obtained f r o m a w o m a n on chronic d i p h e n y l h y d a n t o i n t r e a t m e n t for epilepsy. The c o n t r o l v a l u e s in c u l t u r e w e r e 7 . 2 5 ± 1 . 8 8 (4) n m o l e s / m i n p e r m g p r o t e i n and i n c u b a t i o n for 24 h w i t h d i b u t y r y l cyclic AMP (0.1 raM) and t h e o p h y l l i n e ( 0 . 5 rnM) p r o d u c e d i n c r e a s e d a c t i v i t i e s ( 4 7 . 7 4 ± 2 . 6 4 (4) n m o l e s / m i n p e r m g protein). With t h e e x c e p t i o n o f t y r o s i n e t r a n s a m i n a s e activity w h i c h is i n c r e a s e d b y b o t h cyclic AMP a n d insulin ( B a r n e t t a n d Wicks [ 3 2 ] , Wicks [ 3 1 ] ).
508 these agents, e.g. phosphoenolpyruvate carboxykinase (Girard et al. [30], Wicks [31] ). Studies on the regulation of glucose-6-phosphatase activity in adult rat liver demonstrate a decreased activity after insulin administration {Fisher and Stetten [4] ). The effect of glucagon or cyclic AMP under these circumstances is not clear. In younger animals, insulin (Dawkins [13], Girard et al. [30] ) prevented the normal postnatal increase in activity, thought to be mediated by glucagon and cyclic AMP (Greengard [15], Girard et al. [30] ). These studies suggest that glucose-6-phosphatase activity in the neonatal liver is capable of being controlled by both glucagon/cyclic AMP and insulin. Dibutyryl cyclic AMP markedly stimulates glucose-6-phosphatase activity in explants. This induction of glucose-6-phosphatase in human fetal liver explants is similar but much more dramatic than the 2-fold stimulation observed by Wicks (personal communication) in fetal rat liver explants exposed to 1 mM dibutyryl cyclic AMP plus 0.5 mM theophylline for up to 40 h. This may be the result of species differences, as has been observed for the induction of enzymes in human and rat fetal liver explants (R~iih~i and Schwartz [26], Kirby and Hahn [33,34] ). Human fetal liver contains a relatively high glucose-6-phosphatase activity in comparison to that found in the adult, while the rat fetal liver contains only a very small percentage of that found in the adult. Under similar assay conditions, Gennser et al. [18] found glucose-6-phosphatase activities of approx. 10 nmoles/min per mg protein. Our results in Fig. 1 demonstrate values of 8--28 nmoles/min per mg protein (mean = 17). These values are approx. 25--35% of those reported for normal adult human liver when assayed in a similar manner (i.e. 25--75 nmoles/min per mg protein (van Hoof and Hers [35], Lundquist et al. [36] ). The values obtained in sub-primate species are all approx. 25 nmoles/min per mg protein for the adult activity assayed at 25 ° C, with the fetal value only 5% o f these (Greengard [15], Dawkins [8] ). Insulin was clearly capable of decreasing the glucose-6-phosphatase activity in control explants. However, in tissues preincubated with dibutyryl cyclic AMP, this effect was somewhat less evident. This observation is consistent with the actions of insulin on the regulation of other aspects of glucose metabolism in the human fetal liver. An example is the insulin opposition to the glycogenolytic effect of cyclic AMP (Schwartz, A.L., Riiih~i, N.C.R. and Rail, T.W., unpublished). The inability of insulin to affect hepatic glucose-6-phosphatase activity in fetal rats in utero (Greengard [15] ) may be a result of the low endogenous hepatic glucose-6-phosphatase activity, species differences, or the presence of additional circulating factors in utero. The present study represents to the best of our knowledge, the first report of cyclic AMP and insulin having opposite effects on hepatic glucose-6-phosphatase activity under the same experimental conditions. The present data provide evidence for the regulation of human fetal liver glucose-6-phosphatase activity. Although the adult liver contains appreciable glucose-6-phosphatase activity (Lundquist et al. [36] ), the effects of fasting/ starvation, diabetes or steroid administration have not been examined in man. However, Moses et al. [37] have reported increased hepatic glucose-6-phosphatase activity in a child following corticosteroid administration. Thus all of these results suggest that hepatic glucose-6-phosphatase in the human may be regulated in much the same way as in other species.
509
Acknowledgements The authors acknowledge the excellent technical assistance of Judy Child and are particularly grateful to Dr J. Schlichtkrull of the Novo Research Institute for the gift of insulin and Dr Walter Gall of Merck, Sharp and Dohme for the gift of actinomycin D. This study was supported in part by grants from the Sigrid Juselius Stifelste, Helsinki; the Cleveland Diabetes Fund; and the U.S. Public Health Service grant No. GM-00661-11/-12. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
Langdon, R.G. and Weakley, D.R. (1955) J. Biol. Chem. 214, 167 Weber, G., Singhal, R.L. and Scrivastava, S.K. (1965) Adv. E n z y m e Regul. 3, 475 Ashmore, J., Hastings, A.B., Nesbett, F.B. and Renold, A.E. (1956) J. Biol. Chem. 218, 77 Fisher, C.J. and Stetten, M.R. (1966) Biochim. Biophys. Acta 1 2 1 , 1 0 2 Cahill, G.F., Z o ttu, S. and Earle, A.S. (1957) Endocl~nology 60, 265 Weber, G. and Cantero, A. (1955) Cancer Res. 1 5 , 6 7 9 Dawkins, M.J.R. (1961) Nature 191, 72 Dawkins, M.J.R. (1966) Br. Med. Bull. 22, 27 Mersmann, H.J. (1971) Am. J. Physiol. 220, 1297 Nemeth, A.J. (1954) J. Biol. Chem. 208, 773 Lea, M.A. and Walker, D.G. (1964) Biochem. J. 9 1 , 4 1 7 Ballard, F.J. and Oliver, I.T. (1965) Biochem. J. 95, 191 Dawkins, M.J.R. (1963) Ann. New Y o r k Acad, Sci. 1 1 1 , 2 0 3 Greengard, O. and Dewey, H.K. (1967) J. Biol. Chem. 242, 2986 Greengard, O. (1969) Biochem. J. 115, 19 Villee, C.A. (1953) J. Appl. Physiol. 5, 437 A u ~ i c h i o , S. and Rigillo, N. (1960) Biol. Neonat. 2, 146 Gennser, G., Lundquist, I. and Niisson, E. (1971) Biol. Neonat. 19, 1 Schwartz, A.L. (1972) Biochem. J. 126, 89 R~iih~i, N.C.R., Schwartz, A.L. and Lindxoos, C.M. (1971) Pediatx. Res. 5, 70 ~,' Fiske, C.H. and Su bbaRow, Y. (1925) J. Biol. Chem. 6 6 , 3 7 5 L owry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265 Hartree, E.F. (1972) Anal. Biochem. 4 8 , 4 2 2 Tardmura, T., Nelson, T., Hollingsworth, R.R. and Shephard, T.H. (1971) Anat. Rec. 1 7 1 , 2 2 7 Schwartz, A.L. (1973) s u b m i t t e d to Biochim. Biophys. Acta R~dh~, N.C.R. and Schwartz, A.L. (1973) E n z y m e 15, 330 Villar-Pilasi, C., Lamer, J. and Shen, L.C. (1971) Ann. New Y ork Acad. Sci. 185, 74 E x t o n , J.H. and Park, C.R. (1968) Adv. E n z y m e Regul. 6 , 3 9 1 Mallette, L.E., E x t o n , J.H. and Park, C.R. (1969) J. Biol. Chem. 244, 5713 Girard, J., Caquot, D. and Guillet, I. (1973) E n z y m e 15, 30 Wicks, W.D. (1969) J. Biol. Chem. 244, 3941 Barnett, C.A. and Wicks, W.D. (1971) J. Biol. Chem. 246, 7201 Kirby, L. and Hahn, P. (1973) Pediatr. Res. 7, 75 Kirby, L. and Hahn, P. (1973) Can. J. Biochem. 5 1 , 4 7 6 van Hoof, F. and Hers, H.G. (1966) in Methods in E n z y m o l o g y (Colowick, S. and Kaplan, N., eds), Vol. VIII, p. 525, Academic Press, New York 36 Lun dquist, A., ~ c k e r m a n , P.A. and Schersten, B. (1969) Enz yme Biol. Clin. 10, 8 37 Moses, S.W., Levin, S., Chayoth, R. and Steinitz, K. (1966) Pediatrics 38, 111
Biochimica et Biophysica Acta, 343 ( 1 9 7 4 ) 5 0 0 - - 5 0 9 © Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s
BBA 2 7 4 0 0
EFFECT OF DIBUTYRYL CYCLIC AMP ON GLUCOSE-6-PHOSPHATASE ACTIVITY IN HUMAN FETAL LIVER EXPLANTS
A L A N L. S C H W A R T Z a'*, N I E L S C.R. R~,IH:~ a a n d T H E O D O R E W. R A L L b
aDepartment of Medical Chemistry, University of Helsinki, Helsinki (Finland) and bDepartment of Pharmacology, Case Western Reserve University, Cleveland, Ohio (U.S.A.) (Received D e c e m b e r 7 t h , 1 9 7 3 )
Summary Glucose-6-phosphatase (EC 3.1.3.9) activity in human fetal liver remains constant at 8--28 nmoles/min per mg protein from the 8th week of gestation to at least week 28 and this value is approximately 25--35% of that found in the adult. This enzyme activity was well maintained for 2--3 days in organ culture of fetal liver explants. Incubation with dibutyryl cyclic AMP (0.1 mM) and theophylline (0.5 mM) increased glucose-6-phosphatase activity 4--8-fold within 24 h. Theophylline alone was ineffective, but markedly potentiated the effects of dibutyryl cyclic AMP. This increase in enzyme activity was completely abolished by simultaneous incubation with cycloheximide or actinomycin D. Insulin clearly decreased glucose-6-phosphatase activity in control tissues after 24 h incubation and tended to diminish the elevated glucose-6-phosphatase activity which resulted from pre-incubation with dibutyryl cyclic AMP. The smallest specimen obtained (36 mm crown-rump length = 6 weeks gestation) wa s capable of elevating glucose-6-phosphatase activity more than 3-fold in response to dibutyryl cyclic AMP incubation, suggesting that the human fetal liver has the competence to respond to hormonal agents at a very early stage of development.
Introduction
Since the initial studies demonstrated that adult rat liver glucose-6-phosphatase (EC 3.1.3.9) activity could be increased by steroid administration, fasting/starvation or diabetes (Langdon and Weakley [1]; Weber et al. [2] ), many investigations have examined the ability of various hormones to regulate
* Present address: D e p a r t m e n t of P h a r m a c o l o g y , Case Western Reserve University, Cleveland, Ohio, U.S.A.
501 this activity. Administration of insulin to adult rats was not only capable of decreasing the elevated glucose-6-phosphatase activity associated with diabetes, but was also capable of reducing the glucose-6-phosphatase activity in normal rats in vivo (Ashmore et al. [3], Fisher and Stetten [4] ). Glucagon, however, was ineffective in altering hepatic glucose-6-phosphatase activity in the dog in vivo (Cahill et al. [5] ). Recently much attention has focused on the mechanisms involved in the regulation of this activity by steroid hormones and insulin (see Weber and Cantero [6] ). The development of glucose-6-phosphatase activity in mammalian liver has been documented for a wide variety of species including the rat (Weber and Cantero [6], Dawkins [7] ), mouse (Dawkins [8] ), pig (Mersmann [9] ), guinea pig (Nemeth [10], Lea and Walker [11] ), rabbit (Dawkins [8] ), and lamb (Dawkins [7], Ballard and Oliver [12] ). In all of these species the hepatic glucose-6-phosphatase activity is undetectable until just prior to birth, followed by a dramatic increase in activity in the initial postnatal period surpassing that level found in the adult animal (Dawkins [8], Lea and Walker [11] ). This postnatal increase has been attributed to synthesis of protein de novo (Dawkins [13] ). In addition, Greengard and her associates have suggested that glucagon and cyclic AMP are the primary physiological trigger mechanism since administration of these agents to fetuses in utero causes a premature increase in glucose-6-phosphatase activity (Greengard and Dewey [14], Greengard [15]). Dawkins [13] was able to demonstrate that insulin administration to the newborn rat retarded the normal postnatal increase in activity, although insulin administration to fetuses in utero did not alter the glucose-6-phosphatase activity (Greengard [ 15 ] ). In contrast to those species described above, the human fetal liver contains detectable glucose-6-phosphatase activity by mid-gestation (Villee [16], Aurrichio and Rigillo [17], Gennser et al. [18] ) and at this stage of development is already 25--50% of the level in adult liver. The only other species to exhibit significant glucose-6-phosphatase activity in the fetal liver is the rhesus monkey (Dawkins [8], ). No studies have reported the regulation of this activity in either of these species. The ability of the organ culture system to maintain fetal liver explants in a chemically defined system devoid of circulating factors present in utero for a period of days allows one to examine the control of glucose-6-phosphatase activity by hormonal agents. The present study describes the regulation of glucose-6-phosphatase activity in human fetal liver explants by cyclic AMP. Materials and Methods Materials
All chemicals were of analytical grade and were purchased from Sigma Chemical Co., St. Louis, Mo., U.S.A. except: cycloheximide from Nutritional Biochemicals, Cleveland, Ohio, U.S.A.; actinomycin D from Merck, Sharp and Dohme, Rahway, N.J., U.S.A.; dibutyryl cyclic AMP from Calbiochem, Los Angeles, Calif., U.S.A.; insulin (porcine m o n o c o m p o n e n t lot number MC-S821506) from Novo Industri, Copenhagen, Denmark. The culture medium was a modified Eagle's minimal essential medium in Hank's balanced salt solution (Schwartz [19] ) and was prepared by Orion Oy/Ab, Helsinki, Finland.
502
Tissues and organ culture Tissue was obtained at legal therapeutic abortion by hysterotomy*. A portion of the tissue was rapidly excised and placed into ice-cold culture medium. The remainder was quick frozen in liquid N2. A complete description of the culture system has been published previously (R~iih~i et al. [ 2 0 ] , Schwartz [19] ). Briefly, explants less than 1 mm 3 were placed at the media-gas (02/CO2 ; 95:5, v/v) interface for up to 4 days in culture. All tissues were preincubated for 20--24 h before the experiments began. Additions to the culture medium were made at appropriate times in volumes of less than 0.125 ml (less than 1% of the total volume), so as not to alter the composition of the medium. Furthermore, most agents were prepared in culture medium before addition. At the appropriate times in culture the explants were removed and quick frozen over liquid N2. All samples were stored at --80 ° C until assayed for glucose-6-phosphatase activity. Since the availability of tissue dictated the range of sample sizes examined and since no t w o specimens were obtained at the same time, each series of culture dishes prepared from one tissue had to constitute a complete experiment. Thus, many factors, including the various tissue ages as well as unaccountable variations occurring during pregnancy and surgery, make if difficult to compare absolute values from one culture tissue to another (e.g. dose response curves from t w o tissues demonstrate the same pattern, although the absolute values are different). Glucose-6-phosphatase activity Glucose-6-phosphatase activity was determined as described earlier (Schwartz [19] ) in which a piece of quick-frozen liver or a sample of 25 pooled explants was homogenized in glass-distilled water and a portion of the whole homogenate was incubated in a system containing 40 mM glucose 6-phosphate, 7 mM histidine, 1 mM EDTA, pH 6.5, for 20 min at 37°C. The release of Pi was measured according to Fiske and S u b b a R o w [21]. This procedure was linear as a function of time and amount of homogenate assayed. The homogenate protein concentration was determined according to Lowry et al. [22] as modified by Hartree [23] with bovine serum albumin as a standard. The glucose-6-phosphatase activity is expressed as nmoles Pi released/min per mg protein. Statistical significance is expressed as P values from Student's t-test; values of P > 0.10 are considered n o t significant. Results Glucose-6-phosphatase activity is present in human fetal liver in the smallest specimen examined (60 mm crown-rump length = 8 weeks gestation**) and remains at a b o u t the same level throughout the first two-thirds of gestation (Fig. 1). Earlier reports of glucose-6-phosphatase activity in human fetal liver were confined to few specimens (Villee [16] ) or those greater than 20 weeks
* **
N o n e o f t h e i n v e s t i g a t o r s p a r t i c i p a t e d in t h e d e c i s i o n t o i n t e r r u p t p r e g n a n c y . G e s t a t i o n a l a g e w a s d e t e r m i n e d f ~ o m t h e c r o w n - r u m p l e n g t h n o m o g r a m o f T a n i m u r a et a t . [ 2 4 ] .
503 28" 24-
==-
~
~o-
~. 16o. ~
12-
i ,~
B-
~- ~
~.
"4
~
0 Crown-rump
Length (cm)
Fig. 1. N o r m a l d e v e l o p m e n t o f g l u c o s e - 6 - p h o s p h a t a s e a c t i v i t y in h u m a n fetal liver. Livers w e r e excised f r o m f e t u s e s d e l i v e r e d b y h y s t e r o t o m y a n d w e r e r a p i d l y f r o z e n in liquid N 2. E n z y m e a c t i v i t y w a s l a t e r d e t e r m i n e d f o r e a c h s p e c i m e n . E a c h p o i n t r e p r e s e n t s o n e liver. G e s t a t i o n a l age v a r i e d f r o m 8 w e e k s ( 6 0 m m c r o w n - r u m p l e n g t h ) t o 28 w e e k s .
gestation (Aurrichio and Rigillo [17] ). Recently, Gennser et al. [18] determined glucose-6-phosphatase activity in tissues of 13--23 weeks gestation. The results described in Fig. 1 are consistent with these previous reports, both in terms of relative activities during fetal development and absolute activity values. When human fetal liver explants were placed in organ culture only a small decrease in activity was observed over the initial 48 h in culture (Fig. 2). The addition of dibutyryl cyclic AMP and theophylline at concentrations which 100 '
8O
"I.o j _~
"..,!
40
o
g
=o.
0 0 Hours
in C u l t u r e
Fig. 2. T i m e c o u r s e o f d i b u t y r y l c y c l i c AMP plus t h e o p h y l l i n e o n g l u c o s e - f i - p h o s p h a t a s e a c t i v i t y in h u m a n f e t a l liver e x p l a n t s . E x p l a n t s w e r e p r e p a r e d f r o m a s p e c i m e n o f 1 7 0 m m c r o w n - r u m p l e n g t h . A f t e r 24 h p r e i n c u b a t i o n , d i b u t y r y l cyclic AMP a n d t h e o p h y l i i n e w e r e a d d e d t o s o m e c u l t u r e dishes ( e - - e ) to y i e l d final c o n c e n t r a t i o n s o f 0.1 m M a n d 0 . 5 m M , r e s p e c t i v e l y . No a d d i t i o n w a s m a d e t o t h e c o n t r o l dishes ( o - - o ) . A t t h e a p p r o p r i a t e t i m e s , t h e e x p l a n t s w e r e r e m o v e d a n d q u i c k f r o z e n o v e r l i q u i d N 2. E a c h d e t e r m i n a t i o n w a s p e r f o r m e d o n 2 5 p o o l e d e x p l a n t s . E a c h figure r e p r e s e n t s t h e m e a n + S.E. ( f o u r d e t e r m i n a t i o n s ) . T h e d i b u t y r y l cyclic A M P - c o n t a i n i n g s a m p l e s are s i g n i f i c a n t l y g r e a t e r t h a n t h e c o n t r o l s at all t i m e p e r i o d s e x a m i n e d (P < 0 . 0 1 ) .
504 TABLE I E F F E C T OF T H E O P H Y L L I N E ON D I B U T Y R Y L C Y C L I C A M P - S T I M U L A T E D G L U C O S E - 6 - P H O S P H A T A S E A C T I V I T Y IN H U M A N F E T A L L I V E R E X P L A N T S All tissues w e r e p r e i n c u b a t e d for 24 h, a f t e r w h i c h d i b u t y r y l cyclic AMP a n d / o r t h e o p h y l i i n e was a d d e d . 24 h l a t e r all tissues w e r e r e m o v e d f r o m c u l t u r e . 25 p o o l e d e x p l a n t s w e r e r e q u i r e d f o r e a c h analysis. E a c h figure r e p r e s e n t s t h e m e a n +_ S.E. ( n u m b e r of d e t e r m i n a t i o n s in p a r e n t h e s e s ) . T h e c r o w n - r u m p l e n g t h of this s p e c i m e n w a s 54 r a m . N.S., n o t significant. Agent
Final concentration
T h e ophylline (0.5 m M )
Glucose-6-phosphatase activity ( n m o l e s / m i n p e r mg protein)
None None D i b u t y r y l cyclic AMP D i b u t y r y l cyclic AMP D i b u t y r y l cyclic AMP
--0.1 m M 0.1 m M 0.01 m M
+ + +
17.91 19.54 54.55 70.89 33.94
_+ 4 . 5 5 ± 1.91 + 7.09 -+ 2.25 _+ 5.88
(4) (4) (4) (4) (2)
P
}
n.s.
}
0.03
} ~0.01
were maximal for effects on glycogen metabolism and amino acid uptake (Schwartz [25] ) (0.1 mM and 0.5 mM, respectively) increased glucose-6-phosphatase activity clearly by 8 h after addition (Fig. 2). By 24 h after addition, the activity reached in this experiment was more than 5-fold the control value. Under these same conditions, all livers examined responded in a similar manner with glucose-6-phosphatase activity increases ranging from 3.6-fold (Table I) to 7.7-fold (Table III). In the one experiment in which the dibutyryl cyclic AMP was present for more than 24 h, the glucose-6-phosphatase activity continued to increase at the 48th hour, although this value was not statistically greater than 50-
-
40-
~
.o
3o-. •
¢
20.
o 10-
0
6
.,t o.~o~
o.b~
o'.~ Dibutyryl
Cyclic
~:o AMP
(raM)
Fig. 3. E f f e c t o f d i b u t y r y l cyclic AMP in t h e p r e s e n c e of t h e o p h y l l i n e o n g l u c o s e - 6 - p h o s p h a t a s e a c t i v i t y in h u m a n f e t a l liver e x p l a n t s . E x p l a n t s w e r e P r e p a r e d f r o m a s p e c i m e n of 1 6 5 m m c r o w n - r u m p l e n g t h a n d i n c u b a t e d 24 h w i t h t h e d i h u t y r y l cyclic A M P a f t e r t h e s t a n d a r d p r e i n c u b a t i o n . F o r o t h e r details see t h e l e g e n d t o Fig. 2. E a c h figure r e p r e s e n t s t h e m e a n -+ S.E. ( f o u r d e t e r m i n a t i o n s ) . All d i b u t y r y l cyclic A M P - c o n t a i n i n g s a m p l e s are significantly g r e a t e r t h a n t h e c o n t r o l (P ~ 0 . 0 1 ) . T h e c o n t r o l a n d experim e n t a l dishes c o n t a i n e d 0.5 m M t h e o p h y l l i n e .
505 that after 24 h (P ~ 0.09). These observations demonstrate the marked effect of cyclic AMP as a stimulator of glucose-6-phosphatase activity in human fetal liver. Dose relationships for the cyclic nucleotide were examined and yielded the results depicted in Fig. 3. 0.1 mM dibutyryl cyclic AMP in the presence of theophylline was found to be the maximally effective concentration, with smaller activity increases evident after incubation with either lesser (0.01 mM) or greater (1.0 mM) concentrations of the cyclic nucleotide (Fig. 3). Theophylline alone did not produce marked increases in the glucose-6-phosphatase activity, but potentiated the effects of 0.1 mM dibutyryl cyclic AMP (Table I). This effect is similar to that observed for theophylline effects in human fetal liver explants in examinations of amino acid uptake (Schwartz [25] ) and enzyme induction (R~iihti and Schwartz [26] ). Some insight into the mechanism of the cyclic AMP effect was gained by examining the effects of actinomycin D and cycloheximide on the dibutyryl cyclic AMP-stimulated increase in glucose-6-phosphatase activity. As seen in Table II, addition of actinomycin D alone to the cultures produced only a slight decrease in enzyme activity, when tissues were removed from the cultures 30 h later. However, the simultaneous addition of the antibiotic with the cyclic nucleotide completely prevented the 5-fold increase which resulted from the dibutyryl cyclic AMP alone (Table II). Cycloheximide was added to the cultures at various times after the addition of dibutyryl cyclic AMP. As seen in Table III, preincubations of 0--4 h before cycloheximide addition did not produce increased glucose-6-phosphatase activity. However, there may be a slight increase in activity in tissues preincubated 4 h when compared to the cycloheximide controls (P = 0.03). The effects of insulin were examined on glucose-6-phosphatase activity in these explants. As seen in Table IV, the addition of insulin at the 40th hour in culture significantly decreased the enzyme activity for tissues examined 24 h later. The effect of insulin on dibutyryl cyclic AMP-stimulated enzyme activity was examined by incubating the tissues with maximal concentrations of the cyclic nucleotide plus theophylline for 24 h, after which the culture medium T A B L E II EFFECT OF ACTINOMYCIN D ON DIBUTYRYL CYCLIC AMP-STIMULATED PHATASE ACTIVITY IN HUMAN FETAL LIVER EXPLANTS
GLUCOSE-6-PHOS-
Tissues were p r e i n c u b a t e d f o r 24 h, after w h i c h d i b u t y r y l cyclic A M P , t h e o p h y l l i n e , o r a c t i n o m y c i n D w e r e a d d e d t o y i e l d f i n a l c o n c e n t r a t i o n s of 0.1 m M , 0 . 5 m M a n d 25 ~ g / m l , respectively. The t i s s u e s w e r e r e m o v e d f r o m c u l t u r e 3 0 h l a t e r . 25 p o o l e d e x p l a n t s w e r e r e q u i r e d for each analysis. Each figure represents the m e a n __. S.E. ( n u m b e r o f d e t e r m i n a t i o n s in parentheses). The c r o w n - r u m p length of this sample w a s 95 mm. Agents
h
Glucose-6-phosphatase activity ( n m o l e s / m i n per mg protein)
None Dibutyryl cyclic AMP + theophylline A c t i n o m y cin D D i b u t y r y l cyclic AMP + theophyHine + a c t i n o m y c i n D
30 30 30 30
16.33+ 2.96 (4) 85.02+ 15.14 (4) 10.23+ 0.81 (3) 8.33 __+ 0.95 (4)
P
I} ~
~0.01 0.06 0.09
506 TABLE III E F F E C T O F C Y C L O H E X I M I D E ON D I B U T Y R Y L C Y C L I C A M P - S T I M U L A T E D G L U C O S E - 6 - P H O S P H A T A S E A C T I V I T Y IN H U M A N F E T A L L I V E R E X P L A N T S All tissues w e r e p r e i n e u b a t e d f o r 24 h b e f o r e e x p o s e d to t h e a g e n t s i n d i c a t e d a n d all tissues w e r e r e m o v e d f r o m c u l t u r e a f t e r e x a c t l y 48 h. T h e final c o n c e n t r a t i o n s w e r e 0.1 m M d i b u t y r y l cyclic AMP; 0.5 m M t h e o p h y l l i n e ; 10 # g / m l c y c l o h e x i m i d e . E a c h figure r e p r e s e n t s t h e m e a n ± S.E. ( f o u r d e t e r m i n a t i o n s ) . P v a l u e s are b y c o m p a r i s o n to t h e 24 h c o n t r o l . T h e s p e c i m e n w a s 1 3 5 m m c r o w n - r u m p l e n g t h . Agent
h
Glucose-6-phosphatase activity (nmoles/min per mg protein)
None None D i b u t y r y l cyclic Cycloheximide D i b u t y r y l cyclic + cyeloheximide D i b u t y r y l cyclic + cycloheximide D i b u t y r y l cyclic + cycloheximide D i b u t y r y l cyclic + cycloheximide D i b u t y r y l cyclic + cycloheximide
0 24 24 24 24 24 24 23.5 24 23 24 22 24 20
17.28 10.48 81.05 7.18
AMP + t h e o p h y l l i n e AMP + t h e o p h y l l i n e AMP + t h e o p h y U i n e AMP + t h e o p h y U i n e AMP
+
theophylline
AMP + t h e o p h y l l i n e
± 0.39 + 1.67 +_ 8.51 ± 1.94
P
~0.01 --
~ 0.01 n.s.
9.35 ± 1.28
n.s.
1 0 . 8 6 + 1.42
n.s.
6.10 ± 1.02
0.03
9 . 6 9 _+ 0.27
n.s.
1 2 . 5 4 _+ 1.39
n.s.
T A B L E IV E F F E C T OF C O M B I N A T I O N S OF I N S U L I N A N D C Y C L O H E X I M I D E W I T H D I B U T Y R Y L C Y C L I C AMP ON T H E G L U C O S E - 6 - P H O S P H A T A S E A C T I V I T Y I N H U M A N F E T A L L I V E R E X P L A N T S E x p l a n t s w e r e p r e p a r e d f r o m a s p e c i m e n of 1 0 5 m m c r o w n - r u m p l e n g t h a n d w e r e p r e i n c u b a t e d f o r 16 h in t h e s t a n d a r d m e d i a . A t this t i m e , e i t h e r d i b u t y r y l cyclic AMP ( 0 . I raM) plus t h e o p h y l l i n e (0.5 m M ) or n o a d d i t i o n s w e r e m a d e to t h e dishes. 2 4 h l a t e r s o m e of t h e dishes h a d t h e m e d i a r e p l a c e d w i t h fresh s t a n d a r d m e d i a ( w i t h o u t a g e n t s ) a n d the tissues rinsed. A g e n t s w e r e t h e n again a d d e d t o s o m e dishes (insulin, 1 u n i t / m l ; c y c l o h e x i m i d e , 10 # g / m l ) . E a c h d e t e r m i n a t i o n r ~ q u i r e d 25 p o o l e d e x p l a n t s . E a c h figure r e p r e s e n t s the m e a n + S.E. ( f o u r d e t e r m i n a t i o n s ) . Glucose-6phosphatase activity (nrnoles/min p e r mE p r o t e i n )
Agent added
Glucose-6phosphatase activity (nmoles/min per mg protein)
16 h 17.56 + 1.48
Media wash
Agent added
40 h None
+
None
-
+
Insulin
-
None None
--
+ +
--
+
-
Dibutyryl
54.35
--
AMP
P
64 h
--
cyclic
Gincose-6phosphatase activity (nmoles/min per mg protein)
+ 2.44
11.36+1.43 ) 6.01 -+ 1 . 4 8 8 6 . 1 1 + 21.2 50-58+7.91 l~
Insulin
3 6 79 ± 3 ° 5 S - <
Cycloheximide
3O.68 + 4 . 2 0
<0.02
°:°I
507
was changed, the tissues rinsed in fresh medium and new additions made to the fresh medium. With no new addition the activity was the same when explants were examined 24 h later, perhaps a result of a gradual increase followed by a gradual decrease in activity. Addition of insulin after the medium change tended to decrease the enzyme activity although it was significant only at the P = 0.08 level. Cycloheximide addition after the medium change similarly produced a decrease in activity, perhaps slightly more marked than the insulin effect ( P < 0.05). Re-addition of dibutyryl cyclic AMP plus theophylline further markedly increased the glucose-6-phosphatase activity during the final 24 h of incubation (Table IV). The development of the liver's competence to respond to cyclic AMP by increasing the glucose-6-phosphatase activity was examined in early human fetal livers. The youngest specimen examined was 36 mm crown-rump length (6 weeks gestational age). 24 h incubation with dibutyryl cyclic AMP (0.1 mM) and theophylline (0.5 mM) elevated the control (17.38 + 3.64 (4) nmoles/min per mg protein) activity to 55.32 + 6.69 (4) nmoles/min per mg protein. Younger samples are virtually unobtainable and contain but a few milligrams of tissue. Thus by the 6th gestational week, the fetal liver has the ability to respond to hormonal agents. This same pattern of response has been observed with regard to the ability of the human fetal liver early in gestation (6 weeks) to respond to cyclic AMP in terms of amino acid uptake (Schwartz [25] ) and glycogenolysis (Schwartz, A.L., R~iihR, N.C.R. and Rall, T.W., unpublished) and has been adequately discussed therein*. Discussion
Many of the metabolic parameters involved in the regulation of glucose output by the mammalian liver have been examined in relation to their control by glucagon and cyclic AMP on one hand and insulin on the other. In adult rat liver, glycogenolysis is promoted by glucagon/cyclic AMP (Villar-Pilasi et al. [27] ), whereas insulin stimulates glycogen formation (Exton and Park [28] ). Glucagon/cyclic AMP stimulate gluconeogenesis from [14 C]lactat e or [~ 4C]alanine (Mallette et al. [29] ), while insulin diminishes this effect (Exton and Park [28] ). In addition, urea formation (Exton and Park [28] ) appears to be controlled in opposite directions by these agents. Induction of hepatic enzymes** represents another phenomenon associated with opposite control by
*
**
A specimen ( c r o w n - r u m p length = 65 m m ) was obtained f r o m a w o m a n who had be e n receiving 5 m g o f a s y n t h e t i c p r o g e s t e r o n e / d a y f o r t h e 6 d a y s p r e v i o u s t o a b o r t i o n . The c o n t r o l v a l u e s f o r t h e f e t a l Hver g l u c o s e - 6 - p h o s p h a t a s e a c t i v i t y w e r e w i t h i n t h e n o r m a l r a n g e , as w e r e t h e v a l u e s in c o n t r o l c u l t u r e s ( 2 3 . 2 3 ± 6.41 (4) n m o l e s / m i n p e r m g p r o t e i n ) . H o w e v e r , t h e r e s p o n s e t o 24 h i n c u b a t i o n w i t h d i b u t y r y l cyclic AMP (0.1 m M ) a n d t h e o p h y l i i n e ( 0 . 5 m M ) r e s u l t e d in t h e largest activity observed in a n y tissue u n d e r the s a m e c o n d i t i o n s o f cultuxe ( 1 1 7 . 2 5 + 1 4 . 9 (4) n m o l e s / m i n per mg protein). A second specimen ( c r o w n - r u m p length = 150 ram) was obtained f r o m a w o m a n on chronic d i p h e n y l h y d a n t o i n t r e a t m e n t for epilepsy. The c o n t r o l v a l u e s in c u l t u r e w e r e 7 . 2 5 ± 1 . 8 8 (4) n m o l e s / m i n p e r m g p r o t e i n and i n c u b a t i o n for 24 h w i t h d i b u t y r y l cyclic AMP (0.1 raM) and t h e o p h y l l i n e ( 0 . 5 rnM) p r o d u c e d i n c r e a s e d a c t i v i t i e s ( 4 7 . 7 4 ± 2 . 6 4 (4) n m o l e s / m i n p e r m g protein). With t h e e x c e p t i o n o f t y r o s i n e t r a n s a m i n a s e activity w h i c h is i n c r e a s e d b y b o t h cyclic AMP a n d insulin ( B a r n e t t a n d Wicks [ 3 2 ] , Wicks [ 3 1 ] ).
508 these agents, e.g. phosphoenolpyruvate carboxykinase (Girard et al. [30], Wicks [31] ). Studies on the regulation of glucose-6-phosphatase activity in adult rat liver demonstrate a decreased activity after insulin administration {Fisher and Stetten [4] ). The effect of glucagon or cyclic AMP under these circumstances is not clear. In younger animals, insulin (Dawkins [13], Girard et al. [30] ) prevented the normal postnatal increase in activity, thought to be mediated by glucagon and cyclic AMP (Greengard [15], Girard et al. [30] ). These studies suggest that glucose-6-phosphatase activity in the neonatal liver is capable of being controlled by both glucagon/cyclic AMP and insulin. Dibutyryl cyclic AMP markedly stimulates glucose-6-phosphatase activity in explants. This induction of glucose-6-phosphatase in human fetal liver explants is similar but much more dramatic than the 2-fold stimulation observed by Wicks (personal communication) in fetal rat liver explants exposed to 1 mM dibutyryl cyclic AMP plus 0.5 mM theophylline for up to 40 h. This may be the result of species differences, as has been observed for the induction of enzymes in human and rat fetal liver explants (R~iih~i and Schwartz [26], Kirby and Hahn [33,34] ). Human fetal liver contains a relatively high glucose-6-phosphatase activity in comparison to that found in the adult, while the rat fetal liver contains only a very small percentage of that found in the adult. Under similar assay conditions, Gennser et al. [18] found glucose-6-phosphatase activities of approx. 10 nmoles/min per mg protein. Our results in Fig. 1 demonstrate values of 8--28 nmoles/min per mg protein (mean = 17). These values are approx. 25--35% of those reported for normal adult human liver when assayed in a similar manner (i.e. 25--75 nmoles/min per mg protein (van Hoof and Hers [35], Lundquist et al. [36] ). The values obtained in sub-primate species are all approx. 25 nmoles/min per mg protein for the adult activity assayed at 25 ° C, with the fetal value only 5% o f these (Greengard [15], Dawkins [8] ). Insulin was clearly capable of decreasing the glucose-6-phosphatase activity in control explants. However, in tissues preincubated with dibutyryl cyclic AMP, this effect was somewhat less evident. This observation is consistent with the actions of insulin on the regulation of other aspects of glucose metabolism in the human fetal liver. An example is the insulin opposition to the glycogenolytic effect of cyclic AMP (Schwartz, A.L., Riiih~i, N.C.R. and Rail, T.W., unpublished). The inability of insulin to affect hepatic glucose-6-phosphatase activity in fetal rats in utero (Greengard [15] ) may be a result of the low endogenous hepatic glucose-6-phosphatase activity, species differences, or the presence of additional circulating factors in utero. The present study represents to the best of our knowledge, the first report of cyclic AMP and insulin having opposite effects on hepatic glucose-6-phosphatase activity under the same experimental conditions. The present data provide evidence for the regulation of human fetal liver glucose-6-phosphatase activity. Although the adult liver contains appreciable glucose-6-phosphatase activity (Lundquist et al. [36] ), the effects of fasting/ starvation, diabetes or steroid administration have not been examined in man. However, Moses et al. [37] have reported increased hepatic glucose-6-phosphatase activity in a child following corticosteroid administration. Thus all of these results suggest that hepatic glucose-6-phosphatase in the human may be regulated in much the same way as in other species.
509
Acknowledgements The authors acknowledge the excellent technical assistance of Judy Child and are particularly grateful to Dr J. Schlichtkrull of the Novo Research Institute for the gift of insulin and Dr Walter Gall of Merck, Sharp and Dohme for the gift of actinomycin D. This study was supported in part by grants from the Sigrid Juselius Stifelste, Helsinki; the Cleveland Diabetes Fund; and the U.S. Public Health Service grant No. GM-00661-11/-12. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35
Langdon, R.G. and Weakley, D.R. (1955) J. Biol. Chem. 214, 167 Weber, G., Singhal, R.L. and Scrivastava, S.K. (1965) Adv. E n z y m e Regul. 3, 475 Ashmore, J., Hastings, A.B., Nesbett, F.B. and Renold, A.E. (1956) J. Biol. Chem. 218, 77 Fisher, C.J. and Stetten, M.R. (1966) Biochim. Biophys. Acta 1 2 1 , 1 0 2 Cahill, G.F., Z o ttu, S. and Earle, A.S. (1957) Endocl~nology 60, 265 Weber, G. and Cantero, A. (1955) Cancer Res. 1 5 , 6 7 9 Dawkins, M.J.R. (1961) Nature 191, 72 Dawkins, M.J.R. (1966) Br. Med. Bull. 22, 27 Mersmann, H.J. (1971) Am. J. Physiol. 220, 1297 Nemeth, A.J. (1954) J. Biol. Chem. 208, 773 Lea, M.A. and Walker, D.G. (1964) Biochem. J. 9 1 , 4 1 7 Ballard, F.J. and Oliver, I.T. (1965) Biochem. J. 95, 191 Dawkins, M.J.R. (1963) Ann. New Y o r k Acad, Sci. 1 1 1 , 2 0 3 Greengard, O. and Dewey, H.K. (1967) J. Biol. Chem. 242, 2986 Greengard, O. (1969) Biochem. J. 115, 19 Villee, C.A. (1953) J. Appl. Physiol. 5, 437 A u ~ i c h i o , S. and Rigillo, N. (1960) Biol. Neonat. 2, 146 Gennser, G., Lundquist, I. and Niisson, E. (1971) Biol. Neonat. 19, 1 Schwartz, A.L. (1972) Biochem. J. 126, 89 R~iih~i, N.C.R., Schwartz, A.L. and Lindxoos, C.M. (1971) Pediatx. Res. 5, 70 ~,' Fiske, C.H. and Su bbaRow, Y. (1925) J. Biol. Chem. 6 6 , 3 7 5 L owry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265 Hartree, E.F. (1972) Anal. Biochem. 4 8 , 4 2 2 Tardmura, T., Nelson, T., Hollingsworth, R.R. and Shephard, T.H. (1971) Anat. Rec. 1 7 1 , 2 2 7 Schwartz, A.L. (1973) s u b m i t t e d to Biochim. Biophys. Acta R~dh~, N.C.R. and Schwartz, A.L. (1973) E n z y m e 15, 330 Villar-Pilasi, C., Lamer, J. and Shen, L.C. (1971) Ann. New Y ork Acad. Sci. 185, 74 E x t o n , J.H. and Park, C.R. (1968) Adv. E n z y m e Regul. 6 , 3 9 1 Mallette, L.E., E x t o n , J.H. and Park, C.R. (1969) J. Biol. Chem. 244, 5713 Girard, J., Caquot, D. and Guillet, I. (1973) E n z y m e 15, 30 Wicks, W.D. (1969) J. Biol. Chem. 244, 3941 Barnett, C.A. and Wicks, W.D. (1971) J. Biol. Chem. 246, 7201 Kirby, L. and Hahn, P. (1973) Pediatr. Res. 7, 75 Kirby, L. and Hahn, P. (1973) Can. J. Biochem. 5 1 , 4 7 6 van Hoof, F. and Hers, H.G. (1966) in Methods in E n z y m o l o g y (Colowick, S. and Kaplan, N., eds), Vol. VIII, p. 525, Academic Press, New York 36 Lun dquist, A., ~ c k e r m a n , P.A. and Schersten, B. (1969) Enz yme Biol. Clin. 10, 8 37 Moses, S.W., Levin, S., Chayoth, R. and Steinitz, K. (1966) Pediatrics 38, 111