Effect of aging on induction of rat liver messenger RNA activity for malic enzyme

8iOCHEMlCAL

Vol. 109, No. 1, 1982 November 16, 1982

AND 8lOPHYSlCAL

RESEARCH COMMUNICATIONS Pages 250-255

EFFECT OF AGING ON INDUCTION OF RAT LIVER MESSENGER RNA ACTIVITY Akihiko

Katsurada,

Tezukayama

Nobuko

Iritani,

Hitomi

Tamio

Noguchi*

and Takehiko

Gakuin

College,

Sumiyoshi-ku,

and *Department

Received

FOR MALIC ENZYME

of Nutrition

Osaka

University

September

29,

Medical

Fukuda,

Tanaka* Osaka 558,

and Physiological School,

Kita-ku,

Japan

Chemistry Osaka 530,

Japan

1982

Rats were fasted and then refed a high carbohydrate-fat free diet, and the activities of the mRNA coding for liver malic enzyme [EC 1.1.1.401 in 6-week-old and lo-month-old male rats were determined by in vitro translation of the liver cytoplasmic poly(A)-containing RNA in a rabbit reticulocyte lysate. After refeeding the mRNA activities of the young rats were about 7-fold of those of the aged rats, and roughly parallel to the enzyme activities. This suggests that the age-dependent impairment of the enzyme induction [Iritani, N. et al. (1981) Biochim. Biophys. Acta 665, 6361 can be ascribed to the decrease of mRNA activity. When 1-, diet

were

fed

were

very

high

synthesis

a fat

free

rats

diet,

in l-month-old

rats

analyses

showed

logically

similar

rats

in l-month-old that

the

rats,

to each

other

markedly

with malic

enzyme

decreased

to

different

H.,

Katsurada,

stock

lipogenic

enzymes

The rates

of

and acetyl-CoA 16.1%,

Further,

liversof

(Fukuda,

of

age (1).

respectively. from

to a commercial

induction

and decreased

were

enzymes

adapted of

dehydrogenase,

in g-month-old

of those

previously

the magnitudes

of glucose-6-phosphate

carboxylase 40.8%

2- and g-month-old

18.2%

and

immunochemical

age rats A.,

are

immuno-

mechanism

causing

Iritani.,

submitted). The present the

reductions

cellular that

the

Abbreviation 0006-291 Copyright Ail rights

study of

was initiated

lipogenic

activities

of

the

activity

of malic

to elucidate

enzyme

syntheses

mRNA coding

for

enzyme mRNA is

poly(A)'-RNA:

in aged rats the

Inc. reserved.

250

enzymes.

increased

poly(A)-containing

X/82/210250-06$1.00/0 @ 1982 by Academic Press, of reproduction in any form

a possible

with RNA

by measuring We present the

enzyme

the evidence activity

Vol. 109, No. 1, 1982 by refeeding mRNA level

a fat is

BIOCHEMICAL free

reduced

diet

after

in aged rats

AND

8lOPHYSlCAL

fasting,

and the

as compared

RESEARCH COMMUNICATIONS increase

to young

of

translatable

rats.

MATERIALS AND METHODS Animals Male Wistar rats of 6-week-old and lo-month-old rats were fasted for 2 days and refed a high carbohydrate-fat free diet (1,2) for 3 days. The rats were killed by decapitation, and the livers were quickly removed and immediately frozen with the metal plates chilled with dry ice-acetone. Enzyme purification and antibody preparation Malic enzyme of rat liver was purified essentially according to Hsi and Lardy (3) with a modification. After purification with DEAE-cellulose (Sigma), the enzyme was further chromatographed on an affinity column containing immobilized N6-(6-aminohexyl)adenosine 2',5'-bisphosphate according to Yeung and Carrico (4). The antiserum to rabbit was prepared essentially acmrding to Marshall and Cohen (5). The antibody was purified by affinity chromatography on a protein A-Sepharose Cl-4B column (Sigma) (6). Isolation of poly(A)'-RNA Total cellular RNA was isolated from frozen iiver samples by extraction with 4 M guanidium thiocyanate Fluka purum grade, i Poly(A) -RNA was separated Tridon Inc.) as described by Chirgwin et al. (7). from the total RNA extract by chromatography on oligo(dT) cellulose (Collaborative Research, Type III) (8). Translation of malic enzyme poly(A)'-RNA in reticulocyte lysate system Assays for malic enzyme mRNA activity were carried out with a nucleasetreated reticulocyte lysate system (9). The translation mixture contained 20 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (Hepes)(pH 7.4), 20 ~JM each of 19 amino acids(al1 except methionine), 75 PCi [35S]methionine (lDOO-1260 Ci/mmol; New England Nuclear), various amounts of poly(A)+-RNA and nuclease-treated rabbit reticulocyte lysate in total volume of 80 pl (1D). After incubation at 3O'C for 60 min, the reaction was stopped b{ the addition of 370 ~1 of 20 mM Tris/HCl, pH 7.4 containing lo-5M NADP+, lo- M EDTA, pH 7.4 and 10 mM mercaptoethanol, and centrifuged at 105,000 x g for 60 min. Aliquots of 5 ~1 of the supernatant were taken for determination of radioactivity in total released proteins (Trichloroacetic acid-insoluble materials were determined according to Pelham et al. (9). ). Inununoprecipitation and electrophoresis Malic enzyme was immunoprecipitated from 400 pl of the supernatant with the antibody and formalin-fixed Staphylococcus aureus (Zymed Laboratories) (11,12) in the presence of 0.5% Nonidet P-40, 0.5% desoxycholate and 0.8 mg of rat albumin. Antigen-antibody-bacteria complexes were washed four times with 50 mM Tris/HCl (pH 7.4), 5 mM EDTA, 0.3 M NaCl, 0.5% Nonidet P-40, 0.5% desoxycholate, and 1 mg/ml ovalbumin. Five micrograms of purified malic enzyme was added to the innnunoprecipitates. Then, the immunoprecipitates were dissociated and subjected to electrophoresis on 10% SDS-polyacrylamide slab gels by the method of Laemmli (13). Protein bands were located by staining with Coomassie brilliant blue R250. 3The radioactive bands on slab gels were located by fluorography using EN HANCE (New England Nuclear). The gels were cut into 2 mm wide slices and the radioactivity eluted from the gel slices with NCS tissue solubilizer (Amersham). Radioactivity in each sample was determined in a liquid scintillation spectrometer. 251

Vol. 109, No. 1, 1982

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AND

BIOPHYSICAL

RESEARCH COMMUNICATIONS

Origin-

abcde

v

Fluorogram of dodecylsulfate,$olyacrylamide gel electrophoresis of the ce l-free translation product. [ SlMethionine was incubated with poly(A)'-RNA in a rabbit reticulocyte lysate system. The methioninelabelled translation products were added 0.1 pg of purified malic enzyme and subjected to indirect imnunoprecipitation with antibody. The resulting immunoprecipitates were analyzed as described in Materials and Methods. Lane (a), (b) and (c): inzaunoprecipitates formed with antibody to malic enzyme; 10, 20 and 30 ug/ml of poly(A)'-RNA were used for (a), (b) and (c), resoectively. Lane (d): normal immunoglobulin G replaced the anti-malic enzyme IgG.Lane (e): immunoprecipitate formed with.antibody to malic enzyme 20 ug/ml of poly(A)'-RNA were used for (d) and (e). and 10 pg malic enzyme. The mobility of authentic malic enzyme is indicated to the left of the gels.

RESULTS AND DISCUSSION Poly(A)'-RNA

from

rat

reticulocyte

lysate

The products

isolated

enzyme were

analysed

slab

The fluorogram

gel.

livers

cell-free

was translated

system,

by indirect

in the

in the presence

immunoprecipitation

by electrophoresis is shown

mRNA-dependent of

with

[35S]methionine. antibody

to malic

on a dodecylsulfate/polyacrylamide in Fig. 252

1.

The radioactive

band

in the

Vol. 109, No. 1, 1982

8lOCHEMlCAl

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

10 20 30 PoIY~A~+RNA UI!Urnl)

0

0

[ig. 2 Effects of various amounts of poly(A)+-RNA on 135S methionine incorporation into total protetns and malic enzyme. The 1 4 SS]methionine incorporations in the lane [a), (6) and (.c) of Fig. 1 are shown. The indicated concentrations of Poly(.A)+-RNA were contained in the reaction mixture (80 ~1) to protein s nthesis. Total poly(A)'-RNA activity (0) is expressed as the amount of 13 SS]methionine incorporated into total protein of the reaction mixture. The activity (0) of maTic enzyme poly(A)+-RNA was assayed by measuring the radioactivity band of irmnunoprecipitate formed with antibody to malic enzyme.

immunoprecipitate identical with

to the

Coomassie

parallel

to the

disappearance

the

polypeptide

in the

liver

were

synthesized RNA added for

of

excess

immunoprecipitation

of

the that

2,

the

cell-free

to 20 ug/ml quantitative

radioactive

of

the

When normal

immunoglobulin

no radioactive malic

also

to

the

resulted

G was

translation in the

enzyme.

malic

enzyme

of mRNA for

(stained

seem to be in

to malic

represent

a mobility

band was observed.

enzyme

antibody

Activities

malic

These synthe-

enzyme

in rat

radioactivity.

amount

of malic

system

was proportional

reaction

enzyme

bands

bands

the

with

radioactive

antibody,

with

by measuring

migrated malic

band corresponding

mixture.

in Fig. the

the

radioactive the

of

of unlabelled

to

determined

in

of

enzyme

of authentic

poly(A)'-RNA.

instead

reaction

As shown

to malic

The intensities

amounts

indicated

sized

used

band of

of a large

prior

results

antibody

lysate

The addition product

with

blue).

to the

added

formed

mixture.

purposes. 253

enzyme

as well to the

Therefore,

as total amount this

proteins of

assay

poly(A)'can be

Vol. 109, No. 1, 1982 TABLE I

BIOCHEMICAL

AND

BIOPHYSICAL

RESEARCH COMMUNICATIONS

THE EFFECT OF AGING ON CHANGES IN THE ACTIVITIES OF MALIC ENZYME AND POLY(A)+-RNA CODING FOR MALIC ENZYME OF RAT LIVER DURING FASTING AND REFEEDING Malic enzyme activity

Protein Total

synthesis

protein

Malic

Relative synthesis of malic enzyme to total protein

enzyme

% x 10-2

nmol/min/mg

cpm x 10-5

6-week-old Fasted Refed

20.3 2 1.09 353 5 20.4

5.78 5 0.10 5.22 5 0.43

31 2 5 492 5 41

0.54 2 0.09 9.40 2 0.13

lo-month-old Fasted Refed

18.7 5 3.87 96.1 5 7.85

5.66 2 0.38 7.55 5 2.18

33+ - 8 108 L 30

0.57 5 0.10 1.43 & 0.06

w

Poly(A)+-RNA isolated from the rat livers were incubated with [35S]methioThe translanine at 3O'C for 60 min in a rabbit reticulocyte lysate system. tion mixture contained 20 mM He es (pH 7.4), 20 ~JMeach of 19 amino acids (all 20 pg/ml of the poly(A)'-RNA and except methionine), 75 uCi of [ ! 5S]methionine, The methioninenuclease-treated reticulocyte lysate in total volume of 80 ~1. labelled translation products were separated as shown in Fig. 1 and counted. Malic enzyme activity is expressed as nmol substrate utilized/min/mg of soluble protein at 37OC. Results show mean 5 S.E. (n=3)

Activities changes

in enzyme

to stimulate

translatable

age of

very

low.

In the

is

In the

fasted

those

refed

tively.

Refeeding

17.4-fold

and 2.5-fold

the

than

aged

activities.

rats.

malic

during

(Fukuda,

quantity

rats,

aging

free-high

it

is suggested

The increases

were

can be ascribed due to

the

change

that

that

to the

A.,

age-dependent

decrease

of mRNA activity. 254

in the

of malic were

state

0.094%

rats

in malic

in the young to

rats

enzyme of

immuno-

enzyme activity

N., manuscript

rise

the

concentration

impairment

caused

The activity

parallel

in malic

respec-

fasting

higher

in the

and

and

rats,

after

RNA

enzyme was

respectively.

7-fold

changes

Iritani,

dietary

fasted

roughly

changes

parallel

the

diet

with

of added

and lo-week-old

and aged rats, was about

the

syntheses

corresponding

refeeding

Katsurada,

of

carbohydrate

the young

in parallel

The ability

synthesis

enzyme

to the

enzyme exactly H.,

the

in 6-week-old

We have established

precipitable

induction

of

1.

regardless

the malic

increases

enzyme mRNA after

in the

Therefore,

fat

mRNA varied

in Table

similar

proteins

enzyme mRNA activities of malic

rats,

of total

enzyme

as shown

synthesis

animals.

of

malic

activities,

protein

the

0.014%

of

in submission). in the

in enzyme

enzyme

protein

Vol. 109, No. 1, 1982

BIOCHEMICAL

Correlation

enzyme

for

between

lipogenic

enzymes

dehydrogenase is

reported

al.

(18)

changes

and malic

reported

(19)

hormonal coding

hormonal

this at

the

variation.

translatable

reported change

protein. level

is

the

synthetase,

the

of malic

enzyme

induction

of lipogenic

mRNA activity

reported

6-phosphogluconate

by the mRNA activity

dehydrogenase (20).

enzyme

in neonatal of malic

due to an increase

The responses

has been

Glucose-6-phosphate

levels

The set of

acid

of malic

that

RESEARCH COMMUNICATIONS

and mRNA activity

(14-19).

partly

synthesis

or dietary for

regulated

enzyme

that

also

synthesis

as fatty

to be regulated

in relative

et al.

such

AND BIOPHYSICAL

in response

seem to be reduced

et

mRNA paralleled ducklings.

enzyme

in the enzymes

Siddiqui

Towle

in rat

activity

liver

by

of mRNA

may be coordinately to nutritional with

or

aging.

REFERENCES 1. :: 4. Z: 7. 8. 1;: 11. 12. 13. 14. 15. 16. ii: 19. 20.

Iritani, N., Fukuda, H. and Fukuda, E. (1981) Biochim. Biophys. Acta E, 636-639. Iritani, N. and Fukuda, E. (1980} J. Nutr. E, 1138-1143. Hsi, R. Y. and Lardy, H. A. (1967) J. Biol. Chem. E, 520-526. Yeung, K. K. and Carrico, R. J. (1976) Anal. Biochem.74, 369-375. Marshall, M and Cohen, P. (1961) J. Biol. Chem. 236, 718-724. Goding, J. W. (1978) J. Inununol. Methods 20-, 24lZ3. Chirgwin, J. M., Przybyla, A. E., McDonald, R. J. and Rutter, W. J. (1981) Biochem. a, 5294-5299. Krystosek, A., Cawthon , L. and Kabat, D. (1975) J. Biol. Chem. -'250 6077-6084. Pelham, H. R. B. and Hackson, R. L. (1976) Eur. J. Biochem. 67, 247-256. Noguchi, T. and Tanaka, T. (1982) J. Biol. Chem. z, lllO-li'r3. Kessler, S. W. (1975) J. Immunol. E, 1617-1624. Olson, P. S., Thompson, E. B. and Granner, D. K. (1980) Biochem. l9-, 1705-1711. Laemmli, U. K. (1970) Nature E, 680-685. Nepofroeff, C. M. and Porter, J. W. (1978) J. Biol. Chem. 253, 2279-2283 Flick, P. K., Chen, J., Alberts, A. W. and Vagelos, P. R. m78) Proc. Nat. Acad. Sci. fi, 730-734. Lau, H. P., Nepokroeff, C. M. and Porter, J. W. (1979) Biochem. Biophys. Res. Coaznun. a, 264-271. Hutchinson, J. S. and Holten, D. (1978) J. Biol. Chem. 253, 52-57. Siddiqui, U. A,, Goldflam, T. and Goodridge, A. G. (198rJ. Biol. Chem. E, 4544-4550. Towle, H. C., Maiash, C. M. and Oppenheimer, J. H. (1980) Biochem. B, 579-585. Sun, J. D. and Holten, D. (1978) J. Biol. Chem. E, 6832-6836.

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