J. Nutr.

Metabolism

of Arachidonic, Acids

Sci.

Eicosapentaenoic,

in HepG2

Cells

and

Vitaminol.,

and

Rat

38, 329-334,

1992

Docosahexaenoic

Hepatocytes

Yoko FUJIYAMA-FUJIWARA, Rumi UMEDA, and Osamu IGARASHI Institute of Environmental Science for Human Life, Ochanomizu University, 2-1-1, Ohtsuka, Bunkyo-ku, Tokyo 112, Japan (Received August 23, 1991)

Summary The metabolism of arachidonic acid (AA), eicosapenta enoic acid (EPA), and docosahexaenoic acid (DHA) was examined in HepG2 cells, a human hepatoma cell line, and rat hepatocytes. The AA level in HepG2 cells was lower than in rat hepatocytes and incorporation of AA into HepG2 was also smaller than into rat hepatocytes. Both cells could not increase the level of cellular DHA by the addition of exogenous 22:5 (n-3); whereas, rat hepatocytes, but not HepG2 cells, increased the levels of AA from 20:3 (n-6) and EPA from 20:4 (n-3). In both cells, retroconversion of AA to 20:3 (n-6) occurred, but EPA was not retro converted to 20:4 (n-3). These results suggested that the levels of AA and DHA in both types of cells, were regulated more severely than EPA and that the activity of fatty acid desaturation might be different between n-6 and n-3 families. Key Words HepG2, rat hepatocyte, arachidonic acid, eicosapentaenoic acid, docosahexaenoic acid, desaturation, retroconversion

HepG2 the

cells,

metabolism

known

to

using

cultured

of reduce

metabolism

of

linolenic (8).

of

papers

metabolism

primary

cells acid

cultured

have fatty

rat

acid;

EPA,

been

studied

and

n-3

widely

used fatty

their

roles

(6,7). acid

(EPA)

into in

the

elongation,

and

n-6

docosahexaenoic

in We

for acids

in acid

HepG2 (DHA)

are

compared acid

with

desaturation

series

study

metabolism

previously

cells

to

(PUFAs)

lipoprotein

arachidonic

HepG2

a model

(AA) rat

hepatocytes

and cells.

the and ƒ¿-

retroconver So,

we

in HepG2

studied cells

and

hepatocytes.

AA, PUFA,

and

on of

are

Polyunsaturated

(4,5),

acid

reported acids

of AA,

Abbreviations: hexaenoic

have

line,

and ƒÁ-linolenic

eicosapentaenoic

C22

cell

(1-3). lipids

hepatic

no

and

lipoproteins

linoleic to

C20

hepatoma

plasma

acid But

sion the

a human

arachidonic

polyunsaturated

acid;

EPA,

fatty 329

acid.

eicosapentaenoic

acid;

DHA,

docosa

330

Y. FUJIYAMA-FUJIWARA,

R . UMEDA,

MATERIALS HepG2

cells

described

in the

20:4

(n-3)

Japan.

and

added Both

the

types

incubation, policeman. extracts (8),

of

fatty

fatty

acid

were medium

Cell

lipids

of

Protein

was

were

in

acid

conditions

measured

by

HepG2 1

shows

cells the

metabolize not

(n-3)

into

shown

as

fatty

(n-3)

18:3

20:4

(n-3).

control,

levels

of

and

(n-3)

as

when

(n-3)

of ‡™-6 AA

(n-3)

and

desaturase DHA

in

in

control

fatty

al.

a

rubber

(9).

Lipid

a previous

paper

Then Silar

the

1.0mM. air . After

the

5CP

fatty

capillary

previous

paper

(8)

acid

shown

in

fatty

Table

not cells

rat

24h.

EPA;

acids

were

HepG2

into

(0 .5mM)

2,

whereas but

were

in

37•Ž.

not

added

These

contrast

to

could

both

results

with were

Table

cells

, rat hepatocytes converted 18:4

easily

detected.

hepatocytes

at

HepG2

rat

higher

cells,

suggested hepatocytes

than

.

those

of

HepG2. Figure HepG2 cells

cells

1 shows and

dose-dependently

Table

1.

Control was

Fatty

means

at 0.5mM

the

their

concentration

metabolites and

converted

acid metabolism

no addition

of after

AA,

24-h

of any fatty

EPA,

incubation.

in part

in HepG2

.

(10).

after not

of

and

and

DISCUSSION

but

the

et in

standard.

in

nitrogen

by

Folch

Rascot

al.

the

, 0.75, CO2-95%

method

detail

methods

albumin

harvested of

the

et

various

EPA

0.25

were

with in

under serum

5%

internal

concentration

20:4

But

20:4

an

Lowry

AND

with acid

into

metabolize

activity

The

incubated

cellular 18:3

could

higher

were

as

the

the

, 20:3 (n-6), and Co ., Ltd., Tokyo,

bovine

method

equipped

described

RESULTS

in

by

of

(n-3)

of

cells

the

(17:0)

method

of

24h

and

GLC

were the

for

by

by

evaporated

20%

HCl-methanol

by

18:4

were in

37•Ž

cultured

Petrochemical

concentration

extracted

margaric

and

DHA,

ethanol

removed

measured

which

EPA,

solubilized

at

was

prepared

Idemitsu

the final

using

of

were

column

acids was at

methylated

200nmol

AA, from

incubated

the

contents

(8).

IGARASHI

METHODS

were

donated

medium

cells

were with

paper

these

culture

of

hepatocytes

kindly

residual

to

rat

previous

were

Aliquots

stream

acid

and

AND

and O.

into

22:4

and

DHA AA

(n-6)

was and

incorporated

into

incorporated

into

20:3

(n-6)

. This

cells.

acid

in medium.

Addition

of each

fatty

acid

in medium. J. Nutr.

Sci.

Vitaminol.

PUFA

Table

2.

Control

Fatty

means

was at 0.5mM

METABOLISM

acid metabolism

no addition

IN HEPG2

AND

RAT

HEPATOCYTES

331

in rat hepatocytes.

of any fatty

acid in medium.

Addition

of each

fatty

acid

in medium.

Fig. 1. Metabolism of exogenous AA, EPA, and DHA in HepG2 cells. HepG2 cells were incubated with AA, EPA, or DHA (0.25, 0.75, and 1.0mM, respectively) for 24h. After incubation, fatty acid concentrations in cells were measured by GLC. Columns show the fatty acid concentrations, (_??_, elongated from added fatty acid;_??_, added fatty acid;_??_, retroconverted from added fatty acid).

result suggests the occurrence of chain elongation and retroconversion of AA to keep the regular level of AA and to reduce the excess level of cellular AA. EPA was incorporated dose-dependently and produced only 22:5 (n-3), but was not retro converted into 20:4 (n-3) and the level of DHA did not increase. DHA was also incorporated and only retroconverted into 22:5 (n-3) slightly to decrease the incorporated DHA level. But the amounts of incorporated DHA were the same between the concentrations of 0.75mM and 1.0mM. In rat hepatocytes, shown in Fig. 2, similar results were obtained. Differences from HepG2 cells were as follows: Vol.

38, No.

4, 1992

332

Y. FUJIYAMA-FUJIWARA,

R. UMEDA

, and O.

IGARASHI

Fig. 2. Metabolism of exogenous AA, EPA, and DHA in rat hepatocytes . Hepatocytes were incubated with AA, EPA, or DHA for 24h . Fatty acid concentrations in cells were measured as shown in Fig . 1. Columns show the fatty acid concentrations (_??_, elongated from added fatty acid;_??_ , added fatty acid;_??_, retroconverted from added fatty acid) .

Fig.

3.

The

schema

of PUFA

metabolism

in HepG2

and rat hepatocytes J. Nutr.

Sci.

. Vitaminol.

PUFA

1)

AA

uptake

DHA

was We

into

(8).

as

The

AA

excess

to

addition and

were

20:

in

HepG2 by

types

the

present

and

catalyze

or

into

HepG2

not

HepG2

should n-3

series the

result

fatty

DHA

(n-3)

into

exogenous suggested types

of

addition

will

hepatocytes was

that

the

metabolism

but

also

by

(11)

of

were

well

response known

fatty

acid.

We of

severely not

of the

of

of

the

to

have

acid

the n-3

activity

change

essential

fatty

n-6

of ‡™5-

also

still

continue

n-3

and

n-6

fatty

level

fatty

acids

was

such

dietary

series

or

acids

acids

will

on be

fatty the

catalyzed

In the This

rat

hepato of

Also,

not

fatty

acid

found

the

than

possibility different

by

the

Recently

n-6

dietary

of

results

only

n-6.

and

acid

18:4

excess

These

and

by

we

(8).

cells.

by

series

experiments

HepG2

addition

different

n-3

types

DHA.

DHA.

n-3

AA

metabolism

in

both

as

fatty

n-3

of of

introduced

the

of in

of was

in both

the

accumulate

(n-3)

for

AA

of ‡™5-desaturase

the

level cells

uptake

On

because

be

concentration

medium

22:5

of

the of

the of

not

and

will

since

clarified

and ‡™6-desaturase

activity

will

did

families

effect

and

higher

but

of that

AA

from ƒ¿-linolenate

regulated,

into

examined

desaturation

kind

be

could

cultured

activity

to

hepatocytes.

in

(n-3), higher

retroconverted

cells,

et al.

20:5 of

rat

AA

cells

types

the and

requirement

(n-3)

(n-3)

But in both

Since

20:4

was

of

control

(n-6)

AA.

AA

medium

in of

18:4

and

evidence

rat

the into

acid

AA

amounts In

20:3

hepatocytes

latter, (n-3)

amounts of

(n-3) the

the

18:4

small

to

of

cells ‡™5-desaturase

of ƒÁ-linolenate. rat

small

excess

fatty

If it is assumed

of

of

of

the

this

increase

(n-3).

of

(n-6)

in

But

experiment.

increase

than in

of

rat data

retroconversion

of

small

18:4

types

after ‡™6-desaturation of

DHA

of

desaturation

of

concentration

Christiansen activity

than

20:4

both

20:3

addition

smaller

the

of

smaller

Addition acid

shows

cytes.

on

also

also

or

and

incorporation

the (n-6).

but

desaturation

apparent

by

was

production

of also

metabolism

22:4

decreases

in

2)

reported

accumulation

in this

the (n-6),

cells,

HepG2

excess

caused

cells,

(n-3)

in

previously

the

the

of

observed

HepG2

acids

and

to

only

types

333

than

fatty

introduced

hepatocytes,

catalyze

observed

hepatocytes

production

20:3

regulated,

different.

reported

to

n-3

results

elongation

both

was

higher

incorporation

caused

rat

HEPATOCYTES

1.0mM.

and

medium

of ƒ¿-18:3

AA

the

cells was

be

In

to

these

excess

in

of

AA

n-6

on

chain

addition

is severely

small in

rat

medium

would of

in cells

AA

of

the

in

cells. the

the ‡™5-desaturase

very

and

RAT

twice

up

in

This

3 (n-6),

presence

retroconversion

AND

about

of

AA

cells.

(n-6)

observed

was

3, based

of

of

metabolite,

observed

AA

Fig.

addition types

20:3

HEPG2

metabolism

in

of ƒÁ-linolenate

its

both

hepatocytes

the

shown

both

IN

dose-dependently

summarized

into

of

rat

incorporated

hepatocytes

AA

METABOLISM

rich

diet

different

fats

which

n-6

series

whether or

the same

enzyme.

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J. Nutr.

Sci.

Vitaminol.