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.