The effect of lipids on enzyme levels in beating rat heart cells

Vol. 20, No. 4,1965

BlOCHEMlCAL AtiD RIOPHYSICAL RESEARCH COMMUNICATIONS

TlEEFFETCFLIPIDSCNENZIMELEVELSIN Bl3ATINGRATHgbRTCELLS* Atsulco Fujimoto

and Isaac

Uarary

Laboratory of Nuclear Medicine and Radiation Biology, Department of giophysics and Nuclear Yedicine, and Department of Biological Chemistry, School of Medicine, University of California, Los Angeles, California

ReceivedJuly

2, 1965

During there

the incubation

occurs

oxidation

period

a loss

of beating

(Fujimoto

and liarary,

of beating

and a shift 1964).

a sharp drop in malic and isocitric

rat heart from lipid

adenosine triphosphatase

The shift

to carbohydrate

dehydrogenase and an increase in

A similar

which may be correlated loss in calcium activated

(Ca-ATPase), may be correlated

cessation of beating (Harary et al,

in culture

At the sametime there also occurs

glucose 6-phosphate dehydrogenase activities, with a decrease in oxygen uptalce.

cells

with the observed

1964; Kursmitsu and Uarary, 1964).

in metabolism and loss in ability

to oxidize

palmitate

(Fujimoto and Harary, 1964), together with the loss of function, that lipids

play a central

role in the maintenance of heart cell function.

Many observations indicate

that lipids

respiration

Support for the functional

(Bing, 1965).

derives from the observation that fatty

are an important fuel in heart role of lipids

acids and other lipids

restore beating in cells which have stopped beating in a lipid medium (Harary, Investigation

indicated

also

can deficient

1964; Darary et al, In Dress). of the mechanismof the lipid

effect

has centered

*Supported in part by research grant A-2135 from the National Institute of Health, Public Health Service, and contract AT-04-l-CDN-12 between the U.S. Atomic Energy Conmission and the University of California, Los Angeles, Calif. 456

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 20, No. 4,1965

about three

main areas:

in membrane function; The indications ing since

as a specific

and 3) lipids

are that

lipids

synthesized

ATP

and Slater,

1) lipids

cerns

the effect

which

change in culture.

as they may affect

are not a specific

from any metabolic

This report

1965).

energy source; protein

synthesis.

energy source

for

beat-

source may be used (Rarary

deals with

of serum lipids

2) lipids

the third

on the levels

area listed

and con-

of some of the enzymes

MATERIALSANDMRTRGDS Rat heart stituted for

cells

bovine

were incubated

albumin

the whole serum,

Fetuin

(2g/l)

the culture mental

dishes

in complete

or bovine growth

and the attachment

medium after

The cells

two days for

eight

by scraping,

phosphate

dehydrogenase

albumin plus

medium (Rarary

1964; Ruramitsu Mann Research

and Ca-ATPase,

and liarary, Laboratories

was prepared

from fetal

sonal Coamnnrication). serum and fetal

calf

Cell

calf

Serum lipid serum using

growth

of cells

with

free

cells

were

glucose

(Fujimoto

of fatty

acids

was measured by protein

from

(Goodman, 1957).

from 1:l mixture

cold chloroform-methanol

6-

and Rarary,

serum by the method of Fisher was extracted

to

changes of

Bovine albumin was purchased

and prepared

1963).

by the experi-

dehydrogenase,

were determined

1964).

serum lipids,

the attachment

At intervals,

malic

sub-

and Parley,

were incubated days.

and protein

a medium which

medium was replaced

48 hrs.

harvested

In Press).

(500 mg/l)

using

was added to the medium only for

medium once every

Fetuin

in culture

(Hgrary

(Perof human et al,

determination.

REWLTS The activity culture,

in cells

serum albumin

of Ca-ATPase and malic incubated

instead

in a medium which

of whole serum,

this

medium did not grow or divide.

fall

in activity

of glucose

was partially

6-phosphate

dehydrogenase

(Table

contained I).

dehydrogenase,

which

457

time in bovine

incubated

of serum lipids,

Gn the other rises

with

lipid-free

The cells

In the presence

prevented.

fell,

in the

hand, the level

in growing

cells

losing

Vol. 20, No, 4,1965

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE I Effect

of Serum Lipids

0.50

1.17

--

--

-m

--

--

s-

8,750

14,400

--

MS

8

See text

for

function

experiments

(Xuramitsu

the ratio 6-phosphate

150 in cells

150

conditions

The presence

deficient

197

Cells.

18,000

7

glucose

Heart

11,900

me

medium.

in Cultured

--

6

their

on Enzyme Changes

and Harary,

1964),

also

increased

in the albumin

of serum lipids

slowed

this

increase.

In two other

of the

activities,

specific

grown

in lipid

end of seven

at the

dehydrogenase,

malic

supplemented

media,

dehydrogenase days,

to

was 170 and

and 67 and 30 in lipid

cells.

In the absence in the presence experiment,

of lipids

beating

associated

with

while the

was of interest

ated

by the

lipid

lipid

free

albumin.

of malic

These

loss

stopped

in 4 to 5 days,

was maintained

experiments

at the same time

to the

demonstrate

partially

while

end of the

that

preventing

serum lipids ensynre changes

of function. to see if

deficient

dehydrogenase

the beating

the beating

7 to 8 days.

maintain

It

of lipids

After fell

lipids

medium. five from

Cells

days in this 25,500

458

reverse the changes

could were

incubated

medium

to 11,500

initi-

in a medium with

the specific and glucose

activities 6-phosphate

BIOCHEMICAL AND BlOpHySlCAL RESEARCH COMMUNICATIONS

Vol. 20, No. 4, 1965

o--“-w

\

/ i

i

j DAYS

pig.

1 Enzyme changes in lipid

\\ I’ ‘. r’

i

5 1N

6

i

i

CULTURE

deficient

and lipid

supplemented

media.

The cells were cultured in growth medium with purified lipid (specific rctifree albumin. klalic dehydrogemse 0 l l l l 0 vFty X LO ) and glucose 6-phosphate dehydrogenase v -v were determined at the outset and on the second and fifth day. on the fifth day the beating CY’a stopped. At this time indicated by .the arrow, the atedium was changed to one supplemented with serum lipids at a concentration equivalent to that used in the complete growth mediumwhich contains 20% serum.

dehydrogenase went from 54 to 242,

I).

(Figure

At the sametime the beat-

ing of these cells ceased. At this time the mediumwas changed to one containing

serum lipids

three more days.

plus albumin and the cells were incubated for

The beating was restored within 24 hrs.,

6th, 7th and 8th days, cells were collected termined.

~alic

and on the

and the enzyme activities

de-

dehydrogenase increased smoothly from 11,500 on the

5th day to 18,400 on the 8th day.

Glucose 6-phosphate dehydrogenase

decreased from 242 on the 5th day to 129 on the 8th day. resulting

from

the addition

of senan lipid,

restoring

the levels of the enzymes to the original

are

These changes,

in the direction

of

value in the freshly

isolated heart cells.

The previous observetions (Kuary

459

et al, 1964; Kuremitsu and Karery,

Vol. 2O,No.4,1965

1964)

BIOCtlEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

linking

were

enzyme changes

made in growing

strating

the

in glucose Thus, cells,

growth

cells.

may affect the

cell

of the

link control

synthesis. strates

for

termining enzyme systems, A clear

In most

the enzyme activities

present

of the

structural

of the

and thus beating

may affect

beating

to substances, and kind

to be affected

which is

not

or products

may play

has been

by Schimke synthetase,

by the

level

true

for

related

for

the changes

of the anzymss

460

protein as sub-

role

for

in mmmnalian in Hela

in

to the

in de-

cell

cultures.

cells.

The

and arginase medium.

to the pres-

reaction here. It

of

bacterial

the growth

reported

measured.

the poesi-

The process

may be attributed

directly

the malce-

by acting

a direct

(1964)

of

Such a role

arginlnosuccinase,

enzyme changes is

control

elucidated

of arglnine

Lipids

through

synthesized.

to be operative

reported

the reported

This

which

demonstrated

of argininosuccinate

cases

of enzymes

and repression,

has been

which

time.

cell.

hand,

lipids

of

in the control

On the other

which

that

integrity

may be instrumental

environment

The mechanism

at this

in the medium

stim-

characteristic

possibilities

way they

do not

indicate

as inducers

example

substrates

here

may act

ence of an inducer

not

reported

levels,

number

heart

to a change

In Press).

of substrate

has been

the enzyme.

et al,

activity

Induction

shown

(Harary

of enzpe

conversion

in the

medium

determination

the

synthesis were

to the

cultures.

and not

to the albumin

several

chemical

demon-

and increase

changes

population,

by the maintenance

substances

lipids

dahydrogenase

to the

The results

In this

internal

added

beating

exploring

membranes.

up of the would

We are

observations,

and overgrowth.

acids

known.

the beating

of entry

ble

is not

of function,

were made in non-growing

division

in some way may maintain

heart

and malic

909: of the cell

reinitiate

or loss

The present

may be attributed

and fatty

but

action

lipids

observed

lipids

cultures.

dehydrogenase,

due to cell

Serum

of this

cell

malce up over

in population

“dedifferentiation,”

in Ca-ATPaae

C-phosphate

which

ulate

heart

same loss

the changes

with

is,

catalyzed Lipids however,

by are

BIOCHEMICAL

Vol. 20, No. 4, 1965

AND BIOF’HYSICAL RESEARCH COMMUNICATIONS

possible that the kind of energy metabolism of the cell, comitant and possibly unique distribution

with its

con-

of metabolic intermediates,

may create a chemical enviromaeut necessary for the synthesis of heart proteins.

Such a general phenomenonis similar

to that discussed by

Magasanik (1961) and sumarised by the phrase "catabolite This is a generalization inhibition

of certain

repression".

of what was known as the glucose effect, unrelated enzymes by glucose.

reported here may also be the result

The lipid

of a general effect

acids and other lipids

of heart cells

in culture

repression.

are in someway related

(Darary et al, In Press).

effect

on the internal

chemical environment and may be an example of catabolite Fatty

the

to the beating

The observations

reported here open the way to a more detailed molecular study of the relation of liaids

to heart cell specific

function.

SUMMARY Don-dividing

beating rat heart cells,

incubated in a lipid

free

medium, showeda decrease in malic dehydrogenase and Ca-A!I!Paseand an increase in glucose 6-phosphate dehydrogenase. These changes were associated with a cessation of beating.

Serum lipids,

added to the

medium, prevented the enzyme changes and maintained the beating. lipids

Serum

also restored the beating and reversed the enzyme changes which

were caused by the lipid

deficiency.

Sing, B. J., Physiol. Rev., 45, 171 (1965). Fisher, B. W., Personal Camsunication. Fu.iimo~~l~~.nd Harary. I., Biochim. Biophys. Acta, 86, . Goodmaq D. S,, Science, 125, 1296 (1957). Harary, I., Supplement II to Circ. Res. XIV and xv, 120 (1964). Harary, I. and Parley, B., Dxptl. Cell &es., 29. 451 (1963). Harary, I., Fujimoto; A., and Kuramitsu, Ii., liational Cancer Iimt. Monograph, U, 257 (1964). Harary, I., McCarl, B., and Parley, B., Biochim. Biophys. Acta, IQ Press.

461

Vol. 20, No. 4,19&J

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I. and Slater, S. C., Biochlm. Biophys. Acta, 99, 227 (1965). Kuremiteu, Ii. and Iiarary, I., Biochim. Biophye. Acta, 86. 65 (1964). Megasanik, B., Cold Spring Harbor Sym. on Quant. Biol. XXVI, 249 (1961). Schimke, B. T., National Cancer Inatituta Monograph, I& 197 (1964). Harary,

462