Vol.
177,
No.
June
28,
1991
3, 1991
BIOCHEMICAL
BIOPHYSICAL
AND
RESEARCH
COMMUNICATIONS
1056-1061
Pages
ARACHIDONIC ACID SUPPRESSION OF FATTY ACID SYNTHASE GENE EXPRESSION IN CULTURED RAT HEPATOCYTES Michael Unit
K. Armstrong,
of
William
L.
Performance Enhancement, The Upjohn Company,
Received
April
29,
Blake,
and Steven
D. Clarke
Unit 7921, Bldg. 25, Kalamazoo, MI 49001
Lab 418,
1991
SUMMARY: Rat hepatocytes were maintained in a serum-free, hormonally defined medium supplemented with 50-500 PM albuminbound 2O:l (n-9) vs 20:4 (n-6). The induction of fatty acid synthase mRNA by a mix of insulin/dexamethasone/T3 was inhibited in a dose dependent fashion by 20:4 (n-6). The abundance of P-actin mRNA was not suppressed by 20:4 (n-6). The expression of fatty acid synthase was actually stimulated 2-fold by 2O:l (n-9). It would appear that the in vivo inhibition of fatty acid synthase gene expression by dietary polyunsaturated fatty acids is a specific hepatocelluar event. 0 1991AcademicPress, Inc.
Dietary
(n-6)
inhibit
the
and
(n-3)
expression
of
transcription
(1,2).
unsaturated
fatty
A limitation
of
difficult
to
coding
for
liver
to
hormonal
Utilizing
examine
20:4
(n-6)]
primary
Copyright Ail rights
the
have
of rat
that the
that
acid
in vivo.
$1.50
0 1991 by Academic Press, Inc. of reproduction in any form reserved.
been
may
that
it
present
1056
in
is of genes
response. successfully
gene work
gene
a manner
Rat used
actions
fatty
synthase
(1,2).
inter-organ
govern
polyenoic
hepatocytes
observed
effect
regulation
upon
gene
and mono-
hepatocelluar
a model,
fatty
by inhibiting
hepatocellular
factors
suppressed
is
acid
depends
specific
hypothesis
to that
fatty
culture
such
cultures
comparable 0006-291X/91
the
the
and potently
regulatory
studies
is a specific
examine
enzymes
without
enzymes
primary
selectively
saturated
dietary if
and nutrient
(3-8). to
in
lipogenic
are
in vivo
or
acids
contrast,
acids
lipogenic
cells
a model
In
determine
communicaiton
fatty
as
of expression
was designed acids
[e.g.
expression which
was
in
Vol.
177,
No.
3, 1991
BIOCHEMICAL
AND
MATERIALS
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
AND METHODS
Materials. Collagenase D (EC 3.4.99.5) was obtained from Boehringer Mannheim Biochemicals (Indianapolis, IN). Insulin, Waymouth MB 752/l media containing glutamine, and newborn calf serum were purchased from GIBCO (Grand Island, NY). Tissue culture plates were obtained from Falcon Plastics (Oxnard, CA) Fatty acids were obtained from NuChek Prep (Elysin, MN). The complementary DNAs to fatty acid synthase (pBR 322-FAS-17) and p-actin (pBR-BAl) were provided by M.Magnuson (Vanderbilt University) and L.H. Kedes (Stanford University), respectively. Dexamethasone-hexadrol phosphate was obtained from Organon (West Orange, NJ); triiodothyronine (T3) and all other supplies were purchased from Sigma (St. Louis, MO). Henatocvte isolation ands culture conditions. Male, SpragueDawley rats (210-220 g) were fed a high carbohydrate, fat-free diet for 10 days and then fasted 48 h prior to hepatocyte isolation using a modification of Seglen's procedure (7,9). Cell viability was assessed by trypan blue exclusion (795% exclusion) and cellular ATP concentrations (712 nmoles/106 cells). Hepatocytes (3-4 X lo6 cells/plate) were allowed to attach (4 h) to the culture plates in a medium that contained 4% newborn calf serum (7). After the attachment period, the cell monolayers were incubated in the serum- and hormone-free medium for an additional 24 h. Subsequently the medium was replaced with one containing various concentrations of the hormone mixture (insulin, dexamethasone, T3) and various concentrations of albumin-bound (7) 2O:l (n-9) and 20:4 (n-6) as cited in the figures. The choice of 20:4 (n-6) was based upon our observation that hepatocytes in primary culture have a very limited ability to desaturate and/or elongate 18:2 (n6) which is essential for 18:2 (n-6) inhibition of genes Since (n-9) fatty acids coding for lipogenic proteins (5,lO). do not inhibit fatty acid synthase expression in vivo (1,2), a fatty acid [20:1 (n-9)] of chain length equal to 20:4 (n-6) was chosen for comparative purposes. Northern analvses. The quantity of fatty acid synthase mRNA was determined by Northern analyses and subsequent Total RNA was integration of the radiographic signal (1). extracted with RNAzol (Cinna/Bioteckx, Friendswood, TX) and precipitated as recommended by the manufacturer. RNA (10 Pg) from each tissue was denatured with 2M glyoxal and size fractionated by electrophoresis in a 1.5% agarose gel. After electrophoresis, the RNA was electroblotted onto Zeta Probe NY) and cross-linked to the (Bio-Rad, Rockville Centre, membrane via ultraviolet irradiation (3 min at 1200 pW/cm*). Hybridization and stringency washes were conducted as previously described (1). RESULTS The
induction
mixture
of
repressed only actually
of
did
fatty
acid
synthase
mRNA by the
10 nM insulin/dexamethasone/T3 by 200 PM 20:4
not
inhibit
increased
(n-6).
fatty the
level
In
acid of 1057
was significantly contrast,
synthase fatty
hormonal
2O:l
expression, acid
synthase
(n-9) but mRNA
not
Vol.
177,
No.
BIOCHEMICAL
3, 1991
Table
AND
BIOPHYSICAL
COMMUNICATIONS
RESEARCH
1. Effect of 20:4 (n-6) and 2O:l (n-9) on fatty acid rynthase gene expression in cultured rat hepatocytes
mRNA species and relative Concentration and type of fatty acid
abundance
Fatty acid synthase
6-actin
3.67
1.57
None SO UM 20: 1
7.94
1.61
20:4
5.57
2.51
2O:l
7.50
1.54
20:4
3.02
2.27
to:1
6.38
1.40
20:4
1.76
3.60
100 PM
200 t.rM
500 PM 2O:l
6.82
1.37
20:4
0.89
4.12
Rat hepatocy-tes were maintained in primary culture in a media containing 10 nM insulin, dexamethasone, and T3; plus the albumin-bound fatty acid concentrations cited. Each value is the mean of 2-4 plates (SEMc loom). RNA abundance for fatty acid synthase is expressed as the tctal area for the two transcripts. ANOVA revealed significant inhibition (P
177,
No.
June
28,
1991
3, 1991
BIOCHEMICAL
BIOPHYSICAL
AND
RESEARCH
COMMUNICATIONS
1056-1061
Pages
ARACHIDONIC ACID SUPPRESSION OF FATTY ACID SYNTHASE GENE EXPRESSION IN CULTURED RAT HEPATOCYTES Michael Unit
K. Armstrong,
of
William
L.
Performance Enhancement, The Upjohn Company,
Received
April
29,
Blake,
and Steven
D. Clarke
Unit 7921, Bldg. 25, Kalamazoo, MI 49001
Lab 418,
1991
SUMMARY: Rat hepatocytes were maintained in a serum-free, hormonally defined medium supplemented with 50-500 PM albuminbound 2O:l (n-9) vs 20:4 (n-6). The induction of fatty acid synthase mRNA by a mix of insulin/dexamethasone/T3 was inhibited in a dose dependent fashion by 20:4 (n-6). The abundance of P-actin mRNA was not suppressed by 20:4 (n-6). The expression of fatty acid synthase was actually stimulated 2-fold by 2O:l (n-9). It would appear that the in vivo inhibition of fatty acid synthase gene expression by dietary polyunsaturated fatty acids is a specific hepatocelluar event. 0 1991AcademicPress, Inc.
Dietary
(n-6)
inhibit
the
and
(n-3)
expression
of
transcription
(1,2).
unsaturated
fatty
A limitation
of
difficult
to
coding
for
liver
to
hormonal
Utilizing
examine
20:4
(n-6)]
primary
Copyright Ail rights
the
have
of rat
that the
that
acid
in vivo.
$1.50
0 1991 by Academic Press, Inc. of reproduction in any form reserved.
been
may
that
it
present
1056
in
is of genes
response. successfully
gene work
gene
a manner
Rat used
actions
fatty
synthase
(1,2).
inter-organ
govern
polyenoic
hepatocytes
observed
effect
regulation
upon
gene
and mono-
hepatocelluar
a model,
fatty
by inhibiting
hepatocellular
factors
suppressed
is
acid
depends
specific
hypothesis
to that
fatty
culture
such
cultures
comparable 0006-291X/91
the
the
and potently
regulatory
studies
is a specific
examine
enzymes
without
enzymes
primary
selectively
saturated
dietary if
and nutrient
(3-8). to
in
lipogenic
are
in vivo
or
acids
contrast,
acids
lipogenic
cells
a model
In
determine
communicaiton
fatty
as
of expression
was designed acids
[e.g.
expression which
was
in
Vol.
177,
No.
3, 1991
BIOCHEMICAL
AND
MATERIALS
BIOPHYSICAL
RESEARCH
COMMUNICATIONS
AND METHODS
Materials. Collagenase D (EC 3.4.99.5) was obtained from Boehringer Mannheim Biochemicals (Indianapolis, IN). Insulin, Waymouth MB 752/l media containing glutamine, and newborn calf serum were purchased from GIBCO (Grand Island, NY). Tissue culture plates were obtained from Falcon Plastics (Oxnard, CA) Fatty acids were obtained from NuChek Prep (Elysin, MN). The complementary DNAs to fatty acid synthase (pBR 322-FAS-17) and p-actin (pBR-BAl) were provided by M.Magnuson (Vanderbilt University) and L.H. Kedes (Stanford University), respectively. Dexamethasone-hexadrol phosphate was obtained from Organon (West Orange, NJ); triiodothyronine (T3) and all other supplies were purchased from Sigma (St. Louis, MO). Henatocvte isolation ands culture conditions. Male, SpragueDawley rats (210-220 g) were fed a high carbohydrate, fat-free diet for 10 days and then fasted 48 h prior to hepatocyte isolation using a modification of Seglen's procedure (7,9). Cell viability was assessed by trypan blue exclusion (795% exclusion) and cellular ATP concentrations (712 nmoles/106 cells). Hepatocytes (3-4 X lo6 cells/plate) were allowed to attach (4 h) to the culture plates in a medium that contained 4% newborn calf serum (7). After the attachment period, the cell monolayers were incubated in the serum- and hormone-free medium for an additional 24 h. Subsequently the medium was replaced with one containing various concentrations of the hormone mixture (insulin, dexamethasone, T3) and various concentrations of albumin-bound (7) 2O:l (n-9) and 20:4 (n-6) as cited in the figures. The choice of 20:4 (n-6) was based upon our observation that hepatocytes in primary culture have a very limited ability to desaturate and/or elongate 18:2 (n6) which is essential for 18:2 (n-6) inhibition of genes Since (n-9) fatty acids coding for lipogenic proteins (5,lO). do not inhibit fatty acid synthase expression in vivo (1,2), a fatty acid [20:1 (n-9)] of chain length equal to 20:4 (n-6) was chosen for comparative purposes. Northern analvses. The quantity of fatty acid synthase mRNA was determined by Northern analyses and subsequent Total RNA was integration of the radiographic signal (1). extracted with RNAzol (Cinna/Bioteckx, Friendswood, TX) and precipitated as recommended by the manufacturer. RNA (10 Pg) from each tissue was denatured with 2M glyoxal and size fractionated by electrophoresis in a 1.5% agarose gel. After electrophoresis, the RNA was electroblotted onto Zeta Probe NY) and cross-linked to the (Bio-Rad, Rockville Centre, membrane via ultraviolet irradiation (3 min at 1200 pW/cm*). Hybridization and stringency washes were conducted as previously described (1). RESULTS The
induction
mixture
of
repressed only actually
of
did
fatty
acid
synthase
mRNA by the
10 nM insulin/dexamethasone/T3 by 200 PM 20:4
not
inhibit
increased
(n-6).
fatty the
level
In
acid of 1057
was significantly contrast,
synthase fatty
hormonal
2O:l
expression, acid
synthase
(n-9) but mRNA
not
Vol.
177,
No.
BIOCHEMICAL
3, 1991
Table
AND
BIOPHYSICAL
COMMUNICATIONS
RESEARCH
1. Effect of 20:4 (n-6) and 2O:l (n-9) on fatty acid rynthase gene expression in cultured rat hepatocytes
mRNA species and relative Concentration and type of fatty acid
abundance
Fatty acid synthase
6-actin
3.67
1.57
None SO UM 20: 1
7.94
1.61
20:4
5.57
2.51
2O:l
7.50
1.54
20:4
3.02
2.27
to:1
6.38
1.40
20:4
1.76
3.60
100 PM
200 t.rM
500 PM 2O:l
6.82
1.37
20:4
0.89
4.12
Rat hepatocy-tes were maintained in primary culture in a media containing 10 nM insulin, dexamethasone, and T3; plus the albumin-bound fatty acid concentrations cited. Each value is the mean of 2-4 plates (SEMc loom). RNA abundance for fatty acid synthase is expressed as the tctal area for the two transcripts. ANOVA revealed significant inhibition (P
