Regulation of the synthesis of rat liver serine dehydratase by adenosine 3′, 5′-cyclic monophosphate

Vol. 34, No. 6, 1969

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

REGULATION OF THE SYNTHESIS OF RAT LIVER SERINE DEHYDRATASE BY ADENOSINE 3',5'-CYCLIC MONOPHOSPHATE Jean-Pierre

Jostl,

Division

Abraham

W. Hsie

and H. V. Rickenberg*

of Research, National Jewish Denver, Colorado

Hospital

Received February 6, 1969 Sumnary: The formation of rat liver serine dehydratase was stimulated by the injection of animals with adenosine 3',5'-cyclic monophosphate, epinephrine, or glucagon. The enhanced formation of serine dehydratase was sensitive to actinomyof the formation of serine dehydratase by glucose could be tin D. An inhibition overcome by adenosine 3') 5'-cyclic monophosphate or, more effectively, by its dibutyryl derivative.

The formation of intact acids

or adrenalectomized

or by the

mals fed tase

amino

acids

serine

dehydratase.

tivity

represented

synthesis

glucagon

that

This

showed

is

nephrine

rate

cyclase

(10).

of Biophysics, of Microbiology,

the

induction

were

s h owed that the

(cyclic

intracellular

state

is also

concentration reason

stimulation

University

It

we tested

of the

University

of Colorado

748

this the

sug-

rate

of

effects

well

the effect

School School

of

concentration

of

established

of cyclic

formation

of Colorado

in

dehydratase.

steady AMP).

ac-

investigations

one of the metabolic

intracellular

of

dehydratase

decreased

of serine

to ani-

formation

(.4) confirmed

both

of amino

of serine , dehydra-

of serine

used

livers

of glucose

induced

Recent

of glucose

of degradation

For this

AMP on the

the

feeding

monophosphate the

inhibited

in the

of a mixture

of formation

of the enzyme.

[5,6,7,8,9)

increases

and cyclic

l Department 2 Colorado. Department Colorado.

the

to increase

3',5'-cyclic

adenyl

the

intubation

rate

also

that

can be induced

Administration

techniques

that

of studies

epinephrine

ulating

suggested

immunochemical

on liver

adenosine

increased

D and puromycin

and increased

A series

the

oral

(1,2,3).

--de nova synthesis

and also

dehydratase

by the

of glucagon

blocked

quantitative

gestion

rats

injection

Actinomycin

C3).

which

of the enzyme serine

AMP by stimof both

of serine of Medicine, of Medicine,

epi-

dehydratase. Denver, Denver,

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 34, No. 6, 1969

Our findings trolled

show that

by cyclic

in particular

AMP.

loo-115

days prior at six

nates

prepared

to the hour

aminotransferase

by the Biuret

is

con-

by glucose

AMP (as well

as by

phosphate

(dibutyryl

respectively,

is

tyrosine

led

hydratase

over

fected

whereas

jected

with

using

fetal

derivative

of cyclic

of serine

dehydratase

work

by amino of protein

in Table

intraperitospeed

to Walker

dehydratase

dibutyryl

super-

and Parry

Protein

(ILL),

was meas-

untreated

in the

aminotransferase cyclic

liver

formation.

in organ

that

suggesting

coded

by newly that

measured,

that

749

activity

the

All

of serine

activity

de-

was not af-

in the rats

combination

in-

of cyclic

results

culture

(16).

The dibutyryl

AMP in the

stimulation

cyclic

between

were

induction messenger D, at the

AMP

obtained

5 x 10 -6 and 5 x lo -4

dehydratase

D inhibited

synthesized actinomycin

also

and

Similar

of serine

actinomycin

acids

2 demonstrate

than

At concentrations

on the activity

showed

were

was enhanced

treatments.

maintained

AMP and theophylline, the glucokinase

AMP, and the

not by the other

mono-

purposes

Glucokinase

activity

acids,

3',5'-cyclic

of cyclic

specific

animals.

by amino

adenosine

extracts

AMP was more effective

(1)

for

was determined

(13).

and Pries

of the increase

AMP had no effect

Earlier

dehydratase

For comparative

activities

rat

diet

and high

according

of serine

1.

AMP, dibutyryl but

homogenized

AMP) and a combination

of the

cyclic

to Pitot

protein

and injected

Serine

AMP, N6,02!-

to a significant

and theophylline by Wicks

gl ucokinase

shown in Table

tyrosine

were

(ll),

synthesis

cyclic

that

percent

overnight,

(4).

cyclic

aminotransferase

treatments

on an eight

starved

91311) rats weigh-

California

(15).

of the

epinephrine,

kept

according

reaction

glucagon,

were

previously

et al.

The stimulation

sented

liver

formation

of cyclic

Chatsworth,

The livers

as described

tyrosine

dehydratase

in rat

dehydratase

adm'inistration

experiment,

intervals.

to Goldstein

M cyclic

dehydratase

of serine

by the

The animals

according

synthesis

The inhibition

(Don B Lab Animals,

grams.

neally

ured

of serine

or glucagon).

We used Wistar

five

activity

can be overcome

epinephrine

ing

the

the

in vitro. induction

represented RNA.

of serine the --de novo

The results

concentration

pre-

employed,

cyclic AMP theophylline

15.0 11.0

8.1

15.0

0.05

0.05

Each experimental condition was tested in given by oral intubation (4). The other formed at 0 time and at 6 hours; the rats product formed per hour per mg of protein

3’,5’ plus

N6-02v _ dibutyryl cyclic AMP

5.0

5.0

3',5'

AMP

0.01

Epinephrine

cyclic

0.2

Glucagon

of Serine

+ 0.40

+ 0.60

* 0.20

f 0.20

i 0.50

k 0.40

* 0.05

0.21

0.21

0.17

0.19

0.18

0.17

Glucokinase, NADPH/h/mg

pmoles of protein

574 * 350

350 * 33

220 f 50

149 IL 51

95 f 40

157 k 42

Tyrosine aminotransferase, hydroxyphenylpyruvate/h/mg protein

p,moles of

of the 10 essential amino acids was The intubation and injections were peris expressed as micromoles of reaction

f 0.001

iY 0.001

km0.002

+ 0.001

+ 0.001

2 0.001

Dehydratase

groups of 3-4 rats. The equimolar mixture compounds were injected intraperitoneally. were killed after 12 hours. Enzyme activity in liver extract.

0.83

1.47

0.77

1.19

2.22

2.86

1280

Amino

200

0.11

acids

Synthesis

1

Serine dehydratase, pmoles pyruvate/h mg of protein

of the

Control

Dosage per 100 g body weight mg umoles

Stimulation

Table

Vol. 34, No. 6, 1969

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table Effect

of Actinomycin

2

D on the Synthesis

0.23

N6,02' -dibutyryl

cyclic

AMP

Glucagon

inhibited,

cyclic also

AMP as well

the

as by glucagon.

0.88 t 0.21

0.51

f 0.01

2.09

0.59

A 0.11

+ 0.72

A number

of adenosine

the formation 2',3'

and Pitot

blocks

(3)

by glucose

derepress

The results epinephrine, It

is well

zymes (5); pa=

(.19).

cell

sources

these Both

of serine

in Table

include

cyclic muscle

of these

by cyclic

for

their

ability

to 3',5'

additionalto

the

cyclic

by amino

AMP

to stimAMP, only

in

repeated

AMP overcame

the

repression

AMP affects

the

activity

catalyze glucose.

751

reactions

It

was

AMP would

of glucose.

injections

of glucagon,

by glucose. of a number

and epididymal that

inhibition

cyclic

the presence

delayed

(17,18)

the

Peraino

of glucagon.

or dibutyryl

dehydratase

acids.

as inducer,

concentration

epinephrine

phosphorylase

enzymes

dehydratase

was employed

3 show that

cyclic

known that

of energy

of serine

to see, whether

or dibutyryl

compared

in addition

by increasing

the formation presented

were

when glucagon

be overcome

of interest

induction

by dibutyryl

significantly.

induction

showed that

could

therefore

the

that

dehydratase

D

of protein.

dehydratase;

stimulated

0.1 mg of actinomycin to the injection of represent the average

of serine

suggests

monophosphates

of serine

analogue

induction

This

the -v de novo synthesis

Glucose

also

partially,

reflects

ulate the

albeit

D

2 0.11

The experimental design was as described in Table 1. per 100 g of body weight was injected 30 minutes prior the stimulating agent. The serine dehydratase values of 3 animals.

also

Dehydratase

Serine dehydratase, pmoles pyruvate/h/mg protein actinomycin D plus actinomycin

minus Control

of Serine

make available

of enfat

pad lito the

Vol. 34, No. 6, 1969

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Table 3 The Glucose Effect

and its

Reversal

Serine dehydratase umoles pyruvate h mg protein 1.

Untreated,

2.

Amino acids at 0 time and 6 hrs

3.

4.

animals

killed

at 0 time

0.28

+ 0.0

Animals killed

at 6 hrs

1.61 t 0.65

Animals killed

at 12 hrs

5.65 + 0.42

Amino acids and glucose at 0 hrs and 6 hrs Animals killed

at 6 hrs

0.51 f 0.20

Animals killed

at 12 hrs

0.47 f 0.30

Amino acids and glucose at 0 hrs and 6 hrs Animals killed

at 12 hrs

a)

Glucagon 6 hrs + 9 hrs

1.44 + 0.30

b)

Glucagon + caffeine

1.79 f 0.28

c)

Epinephrine + caffeine

a)

16,02’ -dibutyryl

6 hrs + 9 hrs

cyclic

6 hrs + 9 hrs

1.94 A 0.37

AMP 6 hrs + 9 hrs

1.11

+ 0.08

There were three groups of "control rats" and four groups of rats treated to reverse the inhibition by glucose. (1) Untreated rats (.3 animals) killed at 0 time. (.2) Rats "induced" by the mixture of amino acids. Six rats were intubated with 200 mg (per 100 g of body weight) of the mixture of amino acids at 0 time. Three of the rats were killed after 6 hours. The remaining three animals were given a second dose of amino acids and killed at 12 hours. (,3) Rats in which "induction" inhibited by glucose. Six rats were intubated with the amino acids and 1 g of glucose (.per 100 g of body weight) at 0 time. Three of the rats were killed after 6 hours. The remaining animals were given a second dose of amino acids and glucose and killed at 12 hours. (4) Rats in which inhibition by glucose reversed. Four groups of three rats each were treated with the mixture of amino acids and glucose at 0 time and at 6 hours. At 6 hours and at 9 hours the samegroups of three rats received respectively (,per 100 g of body weight): (a) 0.1 micromole of glucagon, (b) 0.1 micromole of glucagon and 15 micromoles of caffeine, (c) 0.2 micromoles of epinephrine and 15 micromoles of caffeine, and (,a) 6 micromoles of dibutyryl The 12 animals were killed at 12 hours; i.e. cyclic AMP (injected via tail vein). 6 hours after reversal of inhibition by glucose.

There are, to our knowledge, only a few reported cases of the stimulation the synthesis of msmmalianenzymes by cyclic

752

AMPper se.

Yeung and Oliver

(20)

of

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 34, No. 6, 1969

found that cyclic

AMP induced the precocious formation of phosphoenolpyruvate

carboxykinase 3 when injected butyryl fetal

cyclic

Wicks (21) observed that di-

into fetuses -in utero.

AMP induced tyrosine-cu-ketoglutarate

transaminase in explants of

rat liver. The stimulation

of the synthesis of a bacterial

Perlman and Pastan (22) found that in Escherichia co11

been reported recently. the addition

of cyclic

p-galactosidase correlation

AMP to the mediumpartially

formation by glucose.

availability

of cyclic

AMP and

Taken together these findings

AMP may be a key intermediate

of sources of energy to the cell:

of cyclic

of

Janezek et al. (23) observed a positive

of @-galactosidase in E. s.

suggest that in E. coli cyclic

available

overcame the inhibition

between the endogenous steady state concentration

the induclbility

tration

enzyme by cyclic AMP has

that regulates the

the higher the cellular

AMP, the greater the inducibility

concen-

of those enzymes which make

to the bacterium sources of energy additional

to glucose.

The present experiments show that cyclic AMP stimulated and glucose repressed the activity

of serine dehydratase in rat liver.

enzyme and its function

is the introduction

into energy metabolism. role of cyclic

Serine dehydratase is a catabolic

of the amino acids serine and threonine

There may be an analogy between the presumptive regulatory

AMP in the mammaliancell and in E. coli.

actinomycin D partially

inhibited

the increase in serine dehydratase activity

gest, but do not prove, that cyclic enzyme. However earlier

Our experiments in which

AMP stimulated the -de novo synthesis of the

experiments (4) demonstrated conclusively

stimulated the -de novo synthesis of serine dehydratase. ings (5,6,7,8,9,14) cellular

of cyclic

serine dehydratase activity

AMP, it appears likely

tion"

rpay be inappropriate

by increasing the

that the enhancement of

noted in the present work also reflects

synthesis of the enzyme. Furthermore our findings

that glucagon

In view of recent find-

showing that glucagon exerts its effect

concentration

sug-

the -de novo

suggest that the term "induc-

when applied to the stimulation

of the formation of

SThe authors refer to the enzyme as "phosphopyruvate carboxylase"; however the proper nomenclature for the enzymic activity measured is phosphoenolpyruvate carboxykinase.

753

Vol. 34, No.6,

1969

serine dehydratase.

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

"Induction"

in microorganisms implies a steric duced (24). either)

in the case of the induced formation of enzymes relationship

between inducer and the enzyme in-

Usually the inducer is a substrate or a product (or an analogue of

of the reaction

catalyzed by the induced enzyme. No such relationship

exists between serine dehydratase and the substances which stimulate its formation;

the substrates serine and threonine do not induce significantly

the basis of these considerations

and the finding

(2).

that glucose represses the

formation of serine dehydratase we should like to suggest that the regulation serine dehydratase formation may be analogous to "catabolite its

reversal (22) and not to induction,

On

of

repressionM (25) and

sensu stricto. --

Acknowledgments: This work was supported by NIH General Research Support Grant No. FR 05474 to National Jewish Hospital. We should like to thank Miss Ruth Maguire and Mrs. Gerladine Coffey for their competent technical assistance. REFERENCES

1.

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