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Biochem. J. (1969) 115, 19 Printed in Great Britain
The Hormonal Regulation of Enzymes in Prenatal and Postnatal
Rat Liver
EFFECTS OF ADENOSINE 3',5'-(CYCLIC)-MONOPHOSPHATE
BY OLGA GREENGARD
Department of Biological Chemi8try, Harvard Medical School and the Cancer Research In8titute,
New England Deacone88 Hospital, Bo8ton, Mass. 02215, U.S.A.
(Received 18 April 1969)
1. The^ administration of^ glucagon, cAMP [adenosine 3',5'-(cyclic)-monophos-
phate], BcAMP [6-N-2'-O-dibutyryladenosine 3',5'-(cyclic)-monophosphate] or-
adrenaline to foetal rats during the last 2 days of gestation evoked the appearance
of tyrosine aminotransferase and enhanced the accumulation of glucose 6-phos-
phatase in the liver. In foetuses 1-2 days younger only BcAMP was effective.
After birth liver glucose 6-phosphatase no longer responds to glucagon or BcAMP.
Tyrosine aminotransferase is still inducible by these agents in 2-day-old rats, but
not in 50-day-old rats. After adrenalectomy of adults glucagon or BcAMP can
enhance the induction ofthe enzyme by hydrocortisone. The results indicate that the
ability to synthesize tyrosine aminotransferase and glucose 6-phosphatase when
exposed to cAMP develops sooner than the ability to respond to glucagon with an
increase in the concentration of cAMP; the responsiveness of enzymes to different
hormones changes with age. A scheme illustrating the sequential development of
competence in regulating the level of an enzyme is presented. 2. Actinomycin
inhibited the effects of glucagon and BcAMP on liver tyrosine aminotransferase
and glucose 6-phosphatase in foetal rats. Growth hormone, insulin and hydro-
cortisone did not enhance the formation of these enzymes. 3. The time-course of
accumulation of glucose 6-phosphatase in the kidney is different from that in the
liver. Hormones that increase the accumulation in foetal liver do not do so in
the kidney of the same foetus or in the livers of postnatal rats.
Previous studies from our laboratory have shown
that the administration of glucagon to foetal rats
evokes the appearance of tyrosine aminotransferase
(EC 2.6.1.5) and enhances the formation of glucose
6-phosphatase (EC 3.1.3.9) in foetal liver (Greengard
& Dewey, 1967). In the present investigation
adrenaline, growth hormone, insulin and thyroxine
were tested for similar effects on enzymic differenti-
ation in foetal liver and also kidney. An explanation
was sought for the ability of glucagon to evoke
tyrosine aminotransferase in livers of foetuses
during, but not before, the last 2 days of^ gestation.
cAMP,* as briefly reported (Greengard, 1969),
appears to^ be involved in the process whereby
glucagon and^ adrenaline promote the^ developmental
formation of glucose 6-phosphatase and tyrosine
aminotransferase and can evoke these enzymes
earlier. After birth the^ same agents no^ longer
regulate glucose 6-phosphatase; their^ influence^ on
* Abbreviations: cAMP, adenosine 3',5'-(cyclic)-mono-
phosphate; BcAMP, 6-N-2'-O-dibutyryladenosine 3',5'-
(cyclic)-monophosphate.
tyrosine aminotransferase also diminishes with age
and is modified by the endocrine state of the adult
animal.
MATERIALS AND METHODS
Rats were (^) of the (^) Sprague-Dawley CD strain obtained from Charles River (^) Breeding Laboratories, Wilmington, Mass., U.S.A.^ The^ technique of foetal^ injections and^ the enzyme assays were as^ described^ by Greengard &^ Dewey (1967, 1968). Enzyme activities are expressed in units (1 ,umole of product formed/hr. at 25°)/g. wet wt. of tissue. The sources of substances were as follows: hydrocortisone acetate, Merck Sharp & Dohme, West Point, Pa., U.S.A.; growth hormone (0-5 USP unit/mg., Raben type), Nutri- tional Biochemicals Corp., Cleveland, Ohio, U.S.A.;
thyroxine, cAMP and BcAMP, Calbiochem, Los Angeles,
Calif., U.S.A.; glucagon and^ insulin^ (Iletin), Eli^ Lilly and Co., Indianapolis, Ind., U.S.A.; adrenaline (epinephrine- HCI), Parke Davis &^ Co., Detroit, Mich., U.S.A. Substances to be injected into^ foetal rats were^ dissolved^ or^ suspended in 0-1 ml. of 0-9% NaCl soln.; controls received the vehicle only. The doses ofhydrocortisone acetate, cAMP, thyroxine,
growth hormone, glucagon and actinomycin D were 0-125,
O. GREENGARD
0 5, 0 003, 0-25, 0 05 and 001 mg./foetus respectively; the
amount of insulin injected was 0-005 unit. The dose of BcAMP (or AMP) for foetuses above and below 25mm.
body length was 0-25 and 0-125mg. respectively. Postnatal
rats received intraperitoneally 2-5, 0-25 and 2-5mg. of BcAMP, glucagon and hydrocortisone succinate respective- ly/lOOg. body wt.
RESULTS
A series of agents were tested for an effect on the
levels of liver tyrosine aminotransferase, glucose
6-phosphatase and NADPH dehydrogenase (EC
1.6.99.1) in foetal rats 1-2 days before term. Table
1 shows that 5hr. after an injection of adrenaline,
cAMP or BcAMP, foetal livers exhibited significant
tyrosine aminotransferase activity, and that their
glucose 6-phosphatase activity was doubled.
BcAMP, which persists in the tissues longer
(Pasternak, Sutherland & Henion, 1962), was more
effective than cAMP. In contrast, AMP, insulin and
growth hormone were without effect. The NADPH
dehydrogenase activity that was raised by thyroxine
was not raised by BcAMP.
The experiments of Table 2 tested whether un-
inhibited RNA synthesis is required for the induced
rises in the activities of tyrosine aminotransferase
and glucose 6-phosphatase in foetal liver. The results
show that the administration of actinomycin D
prevented the effects of glucagon and of BcAMP on
tyrosine aminotransferase and glucose 6-phospha-
tase. This agent also inhibited the thyroxine-
induced rise in the activity ofglucose 6-phosphatase.
Table 3 compares the effect of glucagon, adren-
aline and BcAMP on liver glucose 6-phosphatase in
rats at different stages of development. In foetal
rats 3-4 days before term, the basal level of
7 5 units, and the 12f3 units reached on an injection
of glucagon, represent small increases over the
substrate-free blank assay values of about 7units.
However, the effect of BcAMP at this age, resulting
Table 1. Induced accumulation of enzymes in foetal rat liver 1-2 day8 (^) before term The indicated substances were administered to individual foetuses (^) intraperitoneally 5hr. before (^) assay unless otherwise indicated. Within each litter some foetuses served as controls (^) (saline). Enzyme activities are (^) expressed
as means+ S.D. of the numbers of observations given in parentheses. -, Not determined.
Enzyme activities (units/g. wet wt.)
Substance injected
Saline Adrenaline cAMP BcAMP BcAMP AMP Insulin Growth hormone Thyroxine
Glucose 6-phosphatase 38+ (^10) (11) 85±9 (4)
40+9 (4) (1hr.)
Tyrosine aminotransferase
<2 (6) (lhr.)
NADP
dehydrogenase 56+10 (^) (13)
45 + 2 (6) (5 or 24hr.)
110+ 19 (18) (24hr).
Table 2. Effect of actinomycin D on induced (^) prenatal enzyme formation in rat liver
Within each of two pregnant rats (about 1 day before term) some foetuses were injected with saline, some
with glucagon and some with actinomycin D plus glucagon. Four other litters were treated similarly, but instead
of glucagon appropriate foetuses of two litters received BcAMP and those of the other two litters received
thyroxine. The^ substances were injected 5hr. before assay. Enzyme activities are expressed as means+S.D.
of the numbers of observations given in parentheses. -, Not determined.
Enzyme activities (units/g. wet wt.)
Tyrosine aminotransferase
Substance (^) injected Saline Glucagon BEAMP Thyroxine
Without actinomycin < 2 (10) 25+3 (5) 15±4 (9)
With actinomycin <2 (^) (6) 2-8_1 (5) < 2 (6)
Glucose (^) 6-phosphatase
Without With actinomycin actinomycin
Table 4. (^) Effect of developmental (^) age on the induced rise in tyrosine aminotransferase activity
in rat liver
Experimental details are similar to those indicated in Table 3. Enzyme activities are expressed as means+ S.D.
of the numbers of observations given in parentheses. -, Not determined. Significance of results :* P < 0*01.
Tyrosine aminotransferase activity (units/g. wet. wt.)
Time before (-) or after (^) (+) birth (days).. ... - 3- Substance (^) injected None < 2 (20)* Glucagon <2 (8) Adrenaline
BcAMP 59 + 1-4 (10)
Hydrocortisone <2 (9)
9-6i4 (6) 4-4±l (5) 11-4_2 (6)
2 (30) 30-0+5 (15) 11±0+5 (4) 15-2+6 (12) <2 (14)
Table 5. Effect of hydrocortisone, glue BcAMP on tyrosine aminotran8feras in adult rat liver The indicated substances, alone or in comi injected 5hr. before assay. Enzyme activities as means+ S.D. of the numbers of observat parentheses. Tyrosine amino
activity (units/
Substance (^) injected
None Hydrocortisone Glucagon BcAMP Glucagon+ hydrocortisone BcAMP+ hydrocortisone
Adrenalectomizec rat 42+3 (20) 220+ 17 (7) 107+22 (6) 97+ 34 (5) 419+ 20 (5) 369+ 36 (4)
a1ult rats the induction oftyrosine amin( by hydrocortisone can be greatly enhai simultaneous administration ofglucagon & (^) Baker, 1966) or adrenaline (Reshef & 1969). BcAMP in adrenalectomized raised the (^) activity of tyrosine amin( from 42 to 97 units (^) (Table 5). The (^) adr of BcAMP together with (^) hydrocortiso in 369units of activity as opposed t obtained with hydrocortisone alone. Thi between hydrocortisone and BcAMP is with glucagon and is not seen in intact ra
DISCUSSION
A series ofobservations indicate that t] ness (^) of agents that promote the de- formation of^ an^ enzyme varies with age a metabolic state or nature of the (^) organ rats 3-4 days before term only BcAMP 4
-agon and the formation of liver tyrosine aminotransferase
e activity and glucose 6-phosphatase whereas in the last
2 days of gestation these effects could also be
obtained with glucagon and adrenaline. Agents
are expressed that^ promoted^ the^ formation of^ glucose 6-phos- ions given in phatase^ in^ foetal^ liver^ did^ not do^ so^ in^ kidneys^ of the (^) same foetuses and did not raise the (^) glucose
transferase 6-phosphatase activity in postnatal livers. Insulin,
/g. wet wt.) growth hormone and hydrocortisone did not
enhance the prenatal formation of tyrosine amino-
I (^) Intact transferase and glucose 6-phosphatase, although rat (^) the latter hormone is an effective inducer of both 60+9 (6) (^) in postnatal rats (Knox & Auerbach, 1955; Weber, 175+54 (6) Singhal, (^) Stamm, Fisher & Mentendiek, 1964). 66+15 (^) (6) (^) Explants of foetal liver cultured in vitro are different 110+15 (^) (3) (^) from both foetal and normal postnatal liver in vivo 169+39 (^) (6) 185+21 in^ that their^ tyrosine^ aminotransferase^ activity (3) can be raised by glucagon, insulin and cAMP as
well as by hydrocortisone (Wicks, 1968a,b).
Adrenalectomy changes the adult rat so that an
otransferase injection of glucagon or BcAMP enhances the
nced by the induction of tyrosine aminotransferase by hydro-
L(Greengard cortisone (Table 5).
Greengard, The developmental formation of liver glucose
adult rats 6-phosphatase consists of distinct prenatal and
otransferase neonatal phases, which are reproducible in foetal
ministration liver^ by the^ separate effects of either^ glucagon (and
)ne resulted also adrenaline and^ cAMP) or^ thyroxine (Greengard
to 220units &^ Dewey, 1968). The other two enzymes studied
is synergism present a simpler picture. Tyrosine aminotrans-
the same (^) as ferase is evoked (^) specifically by glucagon (or
6ts (Table 5). adrenaline or cAMP). NADPH dehydrogenase
responds specifically to (^) thyroxine. Thyroxine does
not evoke tyrosine aminotransferase, and BcAMP
(or glucagon) does not affect NADPH dehydro-.
he effective- genase. (^) These results are consistent with the relopmental possibility that glucagon (or (^) adrenaline) exerts its
ind with the action on tyrosine aminotransferase and glucose
1. In foetal 6-phosphatase through the mediation of cAMP.
could evoke On the other hand, thyroxine does not exert its
22 O.GREENGARD 1969
Vol. 115 DEVELOPMENTAL ENZYME^ FORMATION^23
action on^ NADPH^ dehydrogenase through^ cAMP
(since BcAMP^ is without effect^ on^ this^ enzyme)^ and
does not^ appear to^ raise^ the^ cellular^ concentration
of cAMP (since it does^ not^ evoke^ tyrosine^ amino-
transferase). Glucagon, cAMP and^ BcAMP do not
affect the activity of tyrosine aminotransferase^ or
glucose 6-phosphatase in vitro; the^ rises^ in^ enzyme
activity induced by these agents in vivo^ require
more than an hour^ and^ are^ inhibited^ by^ actino-
mycin D. Thus it is reasonable to assume that the
actions of^ glucagon and^ BcAMP^ now^ studied^ do^ not
consist of^ activations^ of existing enzyme molecules
but increases^ in^ their^ amounts.
The appearance of an enzyme in the normal
course of differentiation must be preceded by a
series of events that permit the function of the
corresponding gene; subsequent changes with age
may eliminate the responsiveness of the enzyme to
certain stimuli and enable it to respond to new
regulators. Scheme 1, illustrating these sequential
events, is based on experience with tyrosine amino-
transferase, but is also relevant (except for the
portion relating to adult animals) to a group of
enzymes that include serine dehydratase, glucose
6-phosphatase and phosphoenolpyruvate carboxyl-
ase. The common features of these enzymes are
that their upsurge is brought about by premature
delivery (Dawkins, 1961; Holt & Oliver, 1968;
Yeung & Oliver, 1968a), that the normal postnatal
rise can be inhibited by the administration ofglucose
(Greengard & Dewey, 1967; Dawkins, 1963; Yeung
& Oliver, 1968b) and that they can be prematurely
induced in foetal liver by glucagon, adrenaline^ or
cAMP (Greengard, 1969; Yeung & Oliver, 1968b).
In Scheme 1 the arrows do^ not^ imply^ mechanism,
but indicate a competence to^ respond to^ the^ stimulus
at the origin of^ the^ arrow with an^ increase^ in^ the
amount of the substance at^ the^ point^ of^ the^ arrow.
For the purposes of^ this scheme BcAMP^ is con-
sidered equivalent to cAMP. The competence to
synthesize tyrosine^ aminotransferase^ under^ the
FOETAL POSTNATAL Age (days) 18 20 22 (last) 14 lo Adult Enzyme Enzyme Enzyme Enzyme Enzyme Actinomycin D -^ -t -----^ -------~~~%*~~~~ ~~~ -------^ ~ t CAMP CAMP CAMP CAMP Cors. Cort.
- Gkucagon Glucagon Glucagon ACTH Glucose --t--------i----s-
- Hypoglycaemia Hypoglycaemais Stress
Scheme 1. Sequential development of competence for regulating an enzyme. ACTH, Adrenocorticotrophic hormone; Cort., glucocorticoids.
influence ofcAMP is present 4 days before birth since
its injection evokes the enzyme at this age. During
the next day or so the capacity develops to raise
the concentration ofcAMP significantly on exposure
to glucagon or adrenaline. Thus the enzyme can be
evoked by these hormones as well as by cAMP. On
the last day before birth the organism is also
competent to respond to hypoglycaemia by the
secretion of these hormones, and therefore pre-
mature delivery at this time can evoke the enzyme.
Finally, the normal newborn animal begins to
synthesize tyrosine aminotransferase because hypo-
glycaemia can stimulate the secretion of the
hormones that raise the concentration of cAMP, a
compound that in turn can initiate the synthesis of
the enzyme. The broken lines in Scheme 1 illustrate
that glucose inhibits the chain of events if given at
birth and that actinomycin D inhibits equally well
the prematurely induced (Table 2) and normal
postnatal (Greengard, Smith & Acs, 1963) accumu-
lation of tyrosine aminotransferase.
Competence in the regulation of tyrosine amino-
transferase continues to change after birth. Within
a day, while the enzyme is still responsive to
glucagon and cAMP, it becomes inducible by hydro-
cortisone. Two weeks later, when the^ pituitary-
adrenocortical axis is^ re-established (Levine &
Mullins, 1966), adrenocorticotrophic hormone^ or
stress can also induce the^ tyrosine aminotransferase
(Knox & Auerbach,^ 1955;^ Schapiro,^ Yuwiler^ &
Geller, 1966). By this^ time^ the^ glucagon 'pathway'
becomes ineffective (Scheme 1, broken arrows) in
normal rats, but is still detectable in adrenalecto-
mized rats (see Table 5). As indicated in Scheme 1,
the mechanism by which glucocorticoids act is
separate from the one involving cAMP. Actino-
mycin D does not distinguish between the two
mechanisms: it inhibits both (Csdnyi, Greengard &
Knox, 1967; see also Table 2). We have no detailed
knowledge of the actual reactions implied by each
arrow in Scheme 1. The scheme simply illustrates
the resolution into sequential steps of the develop-
ment of competence of the liver to synthesize an
enzyme, and to regulate its activity by different
physiological factors at different stages of develop-
ment.
This work was supported by U.S. Public Health Service Grant Ca 07027 from the National Cancer Institute of the National Institutes of Health, and by U.S. Atomic Energy Commission Contract AT(30-1)-3779 with the^ New England Deaconess Hospital.
REFERENCES
Csanyi, V., Greengard, 0. & Knox, W. E. (1967). J. biol. Chem. 242, 2688. Dawkins, M. J. R. (1961). Nature, Lond., 191, 72.
