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INTRODUCTION |
Dietary (n-6) and (n-3) polyunsaturated
fatty acids (PUFA)1 lower
blood triglycerides, decrease intra-muscular lipid droplet size,
improve insulin sensitivity, and enhance nonhepatic glucose utilization
(1-5). PUFA control these metabolic changes in two ways. First, they
induce the transcription of genes encoding proteins involved in lipid
oxidation, e.g. carnitine palmitoyltransferase (6) and acyl-CoA oxidase (7). Second, PUFA suppress the expression of genes encoding proteins involved in lipid synthesis, e.g. fatty acid synthase and acetyl-CoA
carboxylase (8). Genes encoding the oxidative enzymes appear to be
regulated by a common PUFA-activated transcription factor, peroxisome
proliferator-activated receptor
(9, 10). On the other hand, PUFA
appear to coordinately inhibit hepatic lipogenic gene transcription by
rapidly reducing the nuclear content of the lipogenic transcription
factor, sterol regulatory element binding protein-1 (SREBP-1)
(11-14).
There are three members of the SREBP family: 1a, 1c, and 2 (15).
SREBP-1 appears to be more involved with the regulation of lipogenic
genes, while SREBP-2 may have the greatest influence on the expression
of cholesterolgenic genes (16). The SREBPs were identified because of
their ability to bind to the sterol regulatory element and confer
sterol regulation to several genes involved with cholesterol synthesis
(15). SREBPs are synthesized as 125-kDa precursor proteins that contain
two transmembrane domains for insertion into the endoplasmic reticulum
membrane (15). The N-terminal domain, a 68-kDa helix-loop-helix leucine
zipper transcription factor (i.e. mature SREBP), is released
for nuclear translocation by a sterol-dependent proteolytic
cascade (15). The proteolytic release of mature SREBP is also regulated
by PUFA (11-14), but this control may be limited to the release of
SREBP-1. SREBP-1a and -1c are derived from the same gene, but the N
terminus of the SREBP-1a protein is 24 amino acid residues longer
because SREBP-1a and -1c employ different
promoter sites (17).
The nuclear abundance of SREBP-1, and hence the rate of lipogenic gene
transcription, is determined by the rate of proteolytic release of the
mature SREBP-1 and the relative abundance of SREBP-1 precursor
(11-15). The synthesis of SREBP-1 precursor is dependent upon the
relative abundance of SREBP-1 mRNA (11). Fasting,
diabetes, or the ingestion of PUFA reduce the amount of hepatic
SREBP-1 mRNA. On the other hand, carbohydrate-refeeding
or insulin administration increase the abundance of SREBP-1
mRNA (18-20). The changes in hepatic SREBP-1 mRNA
abundance, and ultimately SREBP-1 precursor protein, associated with
fasting, carbohydrate ingestion, diabetes, or insulin administration
reflect alterations in SREBP-1 gene transcription (20).
However, nuclear run-on assays indicated that the reduction in hepatic
content of SREBP-1 mRNA resulting from PUFA ingestion
may involve post-transcription mechanisms (11). This has led us to
hypothesize that PUFA suppress the hepatic expression of
SREBP-1 by accelerating the rate of SREBP-1 mRNA decay. With this report, we in fact demonstrate that PUFA accelerate the decay of SREBP-1 mRNA, and that
SREBP-1c mRNA is more sensitive to PUFA regulation than
is SREBP-1a.
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EXPERIMENTAL PROCEDURES |
Primary Hepatocyte Culture--
Male Harlan Sprague-Dawley rats
(150-170 g) were fasted for 24 h prior to hepatocyte isolation.
Hepatocytes were isolated and maintained in primary monolayer culture
as previously described by Salati and Clarke (21). Briefly, isolated
hepatocytes (9 × 106 cells) were plated onto 10-cm
tissue culture plates that were previously coated with rat tail
collagen (Becton Dickinson Labware). Cells were allowed to attach for
4 h in Waymouth MB 752/1 medium (Life Technology) supplemented
with 0.4 mM alanine, 0.5 mM serine, 26 mM sodium bicarbonate, 100 nM insulin (Life
Technologies Inc.), 100 nM dexamethasone (Sigma), 100 units/ml penicillin, 100 µg/ml streptomycin, and 10% fetal bovine
serum. After the attachment period, medium was changed to a serum-free
media and treated with 150 µM albumin-bound
20:4(n-6), 20:5(n-3), and 18:1(n-9) at
a fatty acid/albumin ratio of 4:1. Cells not treated with fatty acid
received an amount of albumin equal to that provide with the fatty
acid-albumin complex. The source of albumin for all studies was
essentially fatty acid-free bovine serum albumin (Sigma).
Nuclear Run-on Assay and mRNA Analyses--
The impact of
dietary PUFA on the transcription of hepatic fatty acid
synthase and SREBP-1 was determined using the nuclear run-on assay (11). A rat specific SREBP-1c/ADD1 cDNA (B. Spiegelman) was employed to quantify the amount of nascent
SREBP-1 mRNA synthesized by the nuclei. The abundance of
SREBP-1 and fatty acid synthase mRNA in
cultured primary rat hepatocytes was determined using total RNA
extracted by the phenol-guanidinium isothiocyanate procedure (22). For
Northern analysis, total RNA (30 µg per lane) was size-fractionated
on a 1% agarose/formaldehyde denaturing gel, and subsequently
transferred to a Zeta-probe nylon membrane (Bio-Rad) (11). The mRNA
abundance of the respective transcripts was estimated by sequentially
hybridizing the membrane with 32P-labeled cDNA probes
for SREBP-1, fatty acid synthase, and
glyceraldehyde-3-phosphate dehydrogenase. All probes were
labeled with [
-32P]dCTP (PerkinElmer Life Sciences)
using random prime labeling (Life Technologies). Hybridization and wash
conditions have been described previously (23). Autoradiographic
signals were quantified using Instant Imager (Packard). The effect of
fatty acid on the abundance and decay of SREBP-1c and
-1a was determined using the ribonuclease protection assay
(24). A rat SREBP-1a cDNA fragment with sequence
corresponding to exon 1 (specific to SREBP-1a) and part of
exon 2 (common to both SREBP-1a and -1c) was
produced using reverse transcription-polymerase chain reaction
amplification and total rat liver RNA as the template. The primers were
those described in Ref. 17. The amplified cDNA fragment was then
subcloned into the pBluescript vector (Invitrogen) and the plasmid was
linearized with HindIII. The cDNA fragment used to
produce 18S ribosome RNA was purchased from Ambion. Both
antisense probes were transcribed using bacteriophage T3 RNA polymerase
(RPAIII Kit, Ambion). The probes were radiolabeled with
[
-32P]UTP (PerkinElmer Life Sciences) and possessed
specific activities of 5-8 × 108 dpm/µg for
SREBP-1 and 4-10 × 103 dpm/µg for
18S. Hybridization was conducted by incubating 10 µg of
total RNA with at least 4 M excess of probe at 56 °C
overnight. After RNase A/T1 digestion, the protected fragments were
separated on a 8 M urea, 5% polyacrylamide gel. The gel
was dried and the autoradiographic signals were visualized by exposure
to x-ray film, and the abundance of each transcript was quantified
using Instant Imager (Packard). The relative level of
SREBP-1a and -1c mRNA was compared upon
correcting for the number of 32P-labeled UTP atoms in each
protected fragment.
Hepatocyte Abundance of SREBP-1 Protein--
Nuclear and
microsomal protein extracts were prepared from hepatocyte monolayers as
described in Refs. 25 and 26. To prevent proteolysis, all buffers
contained 25 µg/ml N-acetylleucylleucylnorleucinal, 24 µg/ml Pefabloc, 5 µg/ml pepstatin A, 10 µg/ml leupeptin, and 2 µg/ml aprotinin. Briefly, hepatocytes from five 10-cm plates were
pooled, and the cell pellets were disrupted by Dounce homogenization in
3 volumes of a buffer solution containing 10 mM HEPES-KOH
at pH 7.6, 1.5 mM MgCl2, 10 mM KCl,
1 mM sodium EDTA, 1 mM sodium EGTA, 1 mM dithiothreitol and protease inhibitors. The homogenate was centrifuged at 3,300 × g for 15 min. The pellets
were resuspended and incubated for 1 h in an equal volume of a
buffer containing 20 mM HEPES-KOH at pH 7.6, 25% (v/v)
glycerol, 0.5 M NaCl, 1.5 mM MgCl2,
1 mM EDTA, 1 mM EGTA, 1 mM
dithiothreitol and protease inhibitors. The nuclei were collected by
centrifuging the suspension for 30 min at 15,000 × g.
Microsomes were isolated by centrifuging the supernatant from the
initial 3,300 × g spin for 1 h at 100,000 × g. The microsomal proteins were collected by solubilizing
the pellet in 10 mM Tris-HCl at pH 6.8, 0.1 M
NaCl, 1% (v/v) SDS, 1 mM dithiothreitol plus protease
inhibitors. The abundance of mature SREBP-1 (nuclear) and precursor
SREBP-1 (microsomal) was determined by Western blotting following the
procedure described by Xu et al. (11). Immunoreactive
SREBP-1 was identified by incubating the blot for 4 h with
monoclonal anti-SREBP-1 (IgG-2A4) prepared from hybridoma cells (ATCC,
CRL 2121), and the protein visualized using an enhanced
chemiluminescence Western blotting detection system (Amersham Pharmacia
Biotech). Bands were quantified for relative intensity using the Ambis
imagizing system.
Transcription and Translation Inhibition Assays--
PUFA
regulation of SREBP-1a, -1c, and fatty acid
synthase mRNA half-lives in primary hepatocyte was evaluated
by an
-amanitin transcription inhibition assay (27). Hepatocytes
were isolated from fasted rats and maintained in a serum-free media
containing 28 mM glucose, 0.1 µM insulin, and
0.1 µM dexamethasone. Following a 44-h incubation to
induce SREBP-1 expression in the hepatocytes, the medium was
changed to one containing either albumin-bound 150 µM
20:4(n-6) or albumin alone. After a 3-h pretreatment,
transcription was arrested by adding
-amanitin (15 µM
final concentration). The abundance of SREBP-1, fatty acid
synthase, and glyceraldehyde-3-phosphate dehydrogenase
was determined by Northern blot or ribonuclease protection procedures.
To examine the possibility that mRNA translation was needed for
SREBP-1 mRNA to undergo decay, the half-lives of
SREBP-1a and -1c mRNA were determined in the
presence of the translational inhibitor, cycloheximide. As described
previously, SREBP-1 expression in isolated hepatocytes was
induced by culturing hepatocytes in a media containing glucose and
insulin. After 48 h in culture, the cells were treated with
cycloheximide (5 µM final concentration) for 2 h,
and subsequently treated with 150 µM albumin-bound
20:4(n-6) or albumin alone for an additional 6 h. The
abundance of SREBP-1a and -1c mRNA was
quantified using the ribonuclease protection assay.
Cloning of the 3'-Untranslated Region of Rat SREBP-1--
Total
RNA was extracted from rats fed a high carbohydrate diet. The 3'-end of
rat SREBP-1 mRNA was cloned using reverse
transcription-polymerase chain reaction methodology. First strand
synthesis was accomplished using murine leukemia virus reverse
transcriptase and a poly(T) degenerate primer,
5'-TTCTAGTCGACTGAATTCTCTCGAGGCGTTTTTTTTTTTTTTTTTTTT(G/A/C)(G/A/C/T)-3' that was linked to an adaptor sequence of
5'-TTCTAGTCGACTGAATTCTCTCGAGGCG-3'. After a 20-min 42 °C reaction,
polymerase chain reaction amplification was conducted using the
gene-specific primer at position 5'-GAGGAGGGTCTTCCTACATGAGGC-3' and the
adaptor primer sequence 5'-TTCTAGTCGACTGAATTCTCTCGAGGCG-3'. The
reaction conditions were comprised of an initial denaturation at
94 °C for 10 min followed by 5 cycles of 94 °C for 30 s and 72 °C for 1.5 min, 5 cycles of 94 °C for 30 s and 70 °C
for 1.5 min, and finally 33 cycles of 94 °C for 30 s and
68 °C for 1.5 min. The resulting amplification product was sequenced
by the dideoxy chain termination method.
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RESULTS |
SREBP-1 Gene Transcription Is Not Suppressed by
20:4(n-6)--
Earlier work from our group suggested that the hepatic
reduction in SREBP-1 mRNA abundance elicited by dietary
polyunsaturated fatty acids was not accompanied by a reduced rate of
gene transcription (11). However, this earlier work employed a hamster
cDNA for SREBP-1 to quantify the synthesis of nascent
SREBP-1 transcripts that occurred in rat liver nuclei (11).
It is possible that the hamster SREBP-1 cDNA may have
hybridized to a nonspecific nascent transcript produced in the nuclear
run-on assay. Such nonspecific hybridization could have masked
potential changes in SREBP-1 gene transcription. Therefore,
the effect of dietary (n-6) and (n-3)
polyunsaturated fatty acids on rat liver SREBP-1 gene
transcription was re-evaluated using a cDNA specific for rat
SREBP-1, i.e. adipose differentiation
and determination factor-1 (ADD-1). Nuclear
run-on assays (n = 4 per diet group) continued to
indicate that SREBP-1 gene transcription was not inhibited by dietary fats rich in 18-carbon (n-6) or 20- and 22-carbon
(n-3) fatty acids (data not shown).
20:4(n-6) Accelerates SREBP-1 Decay in Isolated
Hepatocytes--
The nuclear run-on results cited above led to the
hypothesis that polyunsaturated fatty acids reduce SREBP-1
mRNA and protein abundance by accelerating the decay of the
SREBP-1 transcript. Pursuit of this question required an
hepatic cellular model that mimicked intact animal responses. Isolated
rat hepatocytes maintained in primary culture fulfill this requirement.
As observed in the intact animal (11-13), the amount of mature SREBP-1
found in the nuclei of hepatocytes was positively correlated with the
hepatocyte content of fatty acid synthase mRNA (Fig.
1). Specifically, culturing hepatocytes
isolated from 24-h fasted rats in medium containing insulin and 28 mM glucose resulted in a 3-4-fold increase in the amount
of membrane (precursor) and nuclear (mature) SREBP-1 (Fig. 1A). The increase in hepatocyte SREBP-1 protein was
paralleled by a comparable rise in the abundance of SREBP-1
mRNA (Fig. 1B). Most importantly, treating the
hepatocytes with 150 µM albumin-bound 20:4(n-6) completely blocked the insulin-glucose dependent
induction of SREBP-1 mRNA and protein (Fig. 1). An
examination of the rapidity with which 20:4(n-6) exerted its
influence on SREBP-1 expression revealed that a 5- and 10-h
exposure to 20:4(n-6) reduced the hepatocyte content of
SREBP-1 mRNA 50 and 85%, respectively (Fig. 2). Interestingly, there was at least a
2-h lag before 20:4(n-6) exerted its suppressive influence
on SREBP-1 mRNA (Fig. 2). Finally, it is important to
note that 20:4(n-6) had no effect on either the
insulin-glucose induction or the steady state level of
glyceraldehyde-3-phosphate dehydrogenase mRNA (Fig.
1B and Fig. 2). This indicates that the suppression of
SREBP-1 and associated lipogenic genes by
20:4(n-6) is specific and comparable to the in
vivo responses achieved with dietary (n-6) or
(n-3) polyunsaturated fatty acids (11-13, 28).

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Fig. 1.
Inhibition of the insulin-glucose induction
of hepatocyte SREBP-1 by
20:4(n-6). Hepatocytes were isolated from 24-h
fasted rats and maintained in a media containing insulin, 28 mM glucose, and 150 µM albumin-bound
20:4(n-6) or 37.5 µM BSA alone. The abundance
of membrane bound (precursor) and nuclear
(mature) SREBP-1 protein (A) was determined by
Western blot analysis using pooled (n = 5 plates)
protein extracts prepared from freshly isolated cells
(initial), or cells treated for 48 h with
20:4(n-6) or BSA. The effect of 20:4(n-6) on
SREBP-1, fatty acid synthase (FAS), and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
mRNA abundance was determined by Northern analysis (B)
using total RNA (30 µg/lane) extracted from hepatocytes immediately
after attachment (initial) or from hepatocytes treated with BSA or
20:4(n-6) for 48 h. The Northern blot is a
representative experiment showing analyses for replicate plates.
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Fig. 2.
The time course for
20:4(n-6) inhibition of SREBP-1
mRNA in rat hepatocytes. Hepatocytes were isolated from
24-h fasted rats and maintained in a media containing insulin and 28 mM glucose. After 44 h in culture (0 time), 37.5 µM BSA or 150 µM albumin-bound
20:4(n-6) was added to the media. Total RNA was extracted
from the hepatocytes at the times indicated, and the abundance of
SREBP-1 and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) mRNA was determined by
Northern analysis (A). B depicts the change in
SREBP-1 mRNA that occurs when hepatocytes were incubated
in the presence (open circles) and absence (closed
circles) of 20:4(n-6). Data are representative of three
experiments and are mean ± S.E., n = 3 plates per
point. Asterisk indicates values that are significantly
lower than the BSA-treated cells.
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The influence of 20:4(n-6) on the half-life of
SREBP-1 mRNA was examined by pretreating rat liver cell
monolayers for 3 h with albumin-bound 20:4(n-6) or
albumin alone, and subsequently adding
-amanitin to inhibit gene
transcription (Figs. 3-5). The abundance
of glyceraldehyde-3-phosphate dehydro-genase was not affected by either 20:4(n-6) or
-amanitin (Figs. 3-5).
On the other hand, Northern analyses revealed that 20:4(n-6)
treatment of rat cell monolayers accelerated the rate of
SREBP-1 mRNA decay (Figs. 3-5). Regression analysis
employing all measurements from 0 to 10 h indicated that
20:4(n-6) significantly (p < 0.05)
shortened the half-life for total SREBP-1 mRNA from
11.6 h (r = 0.94, p < 0.01) in
the absence of fatty acid to 6.4 h (r = 0.92, p < 0.01) in the presence of 20:4(n-6).
Examination of the decay curve suggested that the decay of
SREBP-1 mRNA occurred in two phases, i.e. a rapidly decaying pool (0-4 h), and a more slowly decaying pool (4-10
h). However, 20:4(n-6) accelerated the decay of both of these putative pools (Fig. 3). Because the cDNA probe utilized in
the Northern analyses encoded a sequence that was common to both
SREBP-1a and -1c, we hypothesized that the
apparent existence of two decay rates for SREBP-1, as
determined by Northern analyses, may represent differences in decay
between the SREBP-1a and -1c transcripts. To
examine this possibility, a ribonuclease protection assay was employed
to separately quantify the effect of 20:4(n-6) on the decay
of SREBP-1a and -1c (Fig.
4). The SREBP-1c/1a
ratio found in isolated hepatocytes maintained in a media containing insulin and glucose was 3/1, which was not dissimilar from the 5/1
ratio observed in vivo (29). When hepatocytes were
maintained in a fatty acid-free media, the half-life of
SREBP-1c was similar to that of SREBP-1a,
i.e. 10.0 h (r = 0.77, p < 0.07) and 11.6 h (r = 0.71, p < 0.10), respectively (Fig. 4). However, when the hepatocyte monolayers were treated with 20:4(n-6), the
half-life of SREBP-1c was significantly (p < 0.05) reduced to 4.6 h (r = 0.93, p < 0.01). The half-life of SREBP-1a
mRNA also appeared to be reduced by 20:4(n-6) but the
decrease from 11.6 to 7.7 h (r = 0.94) was not
statistically significant. Like 20:4(n-6), 20:5(n-3) accelerated the decay of SREBP-1c (Fig.
5). The decay of SREBP-1a
appeared to be unaffected by 20:5(n-3), but this lack of
significant decay likely reflects the fact that cells were harvested
after 4 h of fatty acid treatment which was well below the
7.7 h half-life of the SREBP-1a transcript. Unlike the
effect of PUFA, the monounsaturated fatty acid, 18:1(n-9)
had no effect on the hepatocyte content of SREBP-1c and
-1a mRNA. This observation was consistent with several
dietary studies that have shown that SREBP-1 expression is
only suppressed by (n-6) and (n-3)
polyunsaturated fatty acids (8, 11, 23). Finally, the rate of
hepatocyte fatty acid synthase mRNA decay appeared to be
unaffected by either 20:4(n-6) or 20:5(n-3)
(Figs. 3 and 5). Specifically, the half-life of fatty acid
synthase mRNA in monolayers treated with 20:4(n-6) was 5.5 versus 5.8 h for cells treated with BSA alone
(Fig. 3). This observation is consistent with numerous reports
indicating that hepatic fatty acid synthase mRNA levels
are primarily governed by changes in gene transcription (11, 28).

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Fig. 3.
20:4(n-6) accelerated
SREBP-1 mRNA decay in rat liver cell
monolayers. Rat hepatocytes were isolated from 24-h fasted rat
donors and maintained in a media containing insulin and 28 mM glucose. After 44 h in culture, cells were treated
with 150 µM albumin-bound 20:4(n-6)
(open circles) or 37.5 µM BSA alone
(closed circles) for 3 h ( 3) prior to the addition of
the transcription inhibitor, -amanitin. A, the abundance
of SREBP-1, fatty acid synthase (FAS), and
glyceraldehyde-3-phosphate dehydrogenase (GAPDH)
was determined by Northern analysis (30 µg of total RNA per lane).
The logarithmic decay for SREBP-1 and FAS
mRNA are depicted in B and C, respectively.
The SREBP-1 mRNA decay equations for BSA and
20:4(n-6) are y = 0.026x + 1.376 and
y = 0.047x + 1.274, respectively. A test of
differences revealed a significant effect of 20:4(n-6)
(p < 0.05). GAPDH mRNA did not decrease
during the 10-h period. Consequently the abundance of
SREBP-1 and FAS mRNA is expressed relative to
the level of GAPDH. The regression equations for
FAS mRNA decay are y = 0.026x + 1.655 and y = 0.04x + 1.636 for the BSA and
20:4(n-6) treated cells, respectively. Data are
representative of two independent experiments.
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Fig. 4.
20:4(n-6) differentially
regulates transcript decay for SREBP-1a and
SREBP-1c. Using the same RNA samples analyzed in
Fig. 3, ribonuclease protection assays were conducted to quantify the
rate of SREBP-1c and -1a decay (A)
depicts a representative assay. The change in SREBP-1c
(B) and SREBP-1a (C) mRNA
half-life observed in hepatocytes treated with 150 µM
20:4(n-6) or 37.5 µM BSA was calculated and
plotted using the curve-fit of a linear plot. Values were normalized to
the abundance of 18S ribosomal RNA. The regression equations
for SREBP-1c mRNA decay are y = 0.066x + 1.036 versus y = -0.030x + 1.124 for
20:4(n-6) and BSA, respectively (p < 0.05); and the
regression equations for SREBP-1a mRNA decay are
y = 0.04x + 1.636 versus y = 0.026x + 1.655 for 20:4(n-6) and BSA, respectively
(p > 0.05).
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Fig. 5.
The effects of different unsaturated
fatty acids on mRNA decay of SREBP-1c and
SREBP-1a in rat hepatocytes. A, total
RNA was extracted from rat hepatocyte monolayers that had been
pretreated with 250 µM albumin-bound 18:1
(n = 9) (black bars), 20:4(n-6)
(gray bars), or 20:5(n-3) (white bars)
for 3 h. After the 3-h pretreatment period (0 h), -amanitin was
added to the media to halt transcription, and the relative abundance of
SREBP-1c and -1a mRNA was determined by the
ribonuclease protection procedure. Values are expressed relative to
18:1(n-9) at 0 h, and the mRNA values were
normalized to 18S ribosomal RNA. Asterisk denotes
a significant (p < 0.05) reduction from 0 h;
B depicts a representative ribonuclease protection assay for
SREBP-1a and 1c; and C is a
representative Northern blot demonstrating that the decay of
fatty acid synthase (FAS) and
glyceraldehyde dehydrogenase (GAPDH) mRNA was
not affected by 20:4(n-6) or 20:5(n-3).
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Decay of SREBP-1c mRNA Requires Translation--
In an attempt
to ascertain if the PUFA enhancement of hepatic SREBP-1
mRNA decay involves a short-half-life regulatory protein, the
influence of 20:4(n-6) on SREBP-1a and
-1c mRNA decay was examined in the presence of the
translational inhibitor, cycloheximide. Interestingly, cycloheximide
treatment of the hepatocyte monolayers increased hepatocyte
SREBP-1c and -1a mRNA abundance (Fig.
6). Moreover, blocking mRNA
translation completely eliminated the PUFA-dependent
increase in SREBP-1c and -1a mRNA decay (Fig.
6).

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Fig. 6.
Protein synthesis is required for the
decay of SREBP-1. After 44 h in culture,
hepatocytes were treated with cycloheximide (CHX) for 2 h prior to the addition of 150 µM albumin-bound
20:4(n-6) or 37.5 µM albumin alone
(BSA). SREBP-1a and -1c mRNA
abundance was determined 6 h after the addition of
20:4(n-6). A depicts the relative level of
SREBP-1c and -1a mRNA in the absence
(black bars) or presence (white bars) of
20:4(n-6) and with or without cycloheximide. The abundance
of SREBP-1a and -1c mRNA is expressed
relative to 18S mRNA and represent the average of
n = 3 plates per treatment. Asterisk (*)
indicates 20:4(n-6) significantly (p < 0.01) reduced SREBP-1c mRNA; double asterisk
(**) indicates a significant (p < 0.05) increase in
SREBP-1c mRNA due to CHX treatment; B, representative
ribonuclease protection assay for SREBP-1a and
-1c.
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Cloning and Sequence of the 3'-Untranslated Region of Rat
SREBP-1--
Future pursuit of the mechanisms by which PUFA regulate
the decay rate of SREBP-1 mRNA require knowledge of the
complete transcript sequence for rat SREBP-1. However, the
sequence for rat SREBP-1 (i.e. ADD-1)
that was available did not include a termination codon nor did it
include the 3'-untranslated region of the rat SREBP-1
transcript. Using total rat liver RNA, and reverse transcription and
polymerase chain reaction amplification, we successfully cloned a
692-base pair cDNA for rat SREBP-1 that included a stop
codon, a poly(A) tail, and a 350-base pair 3'-untranslated region (Fig. 7). Alignment of the rat SREBP-1
transcript sequence with that for human SREBP-1
revealed that the 3'-end of the rat SREBP-1 protein is highly
homologous with the human, and that the reported sequence for
ADD-1 (i.e. rat SREBP-1) lacked
sequence for 86 amino acid residues (Fig. 7). The 3'-untranslated
region contained four A-U regions that may be candidates for the
regulation of SREBP-1 mRNA stability.

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Fig. 7.
Nucleotide sequence for the 3'-end of
rat SREBP-1 mRNA. The 3'-end of rat
SREBP-1 (rSREBP-1) was cloned as described under
"Experimental Procedures." The nucleotide sequence and the
predicted amino acid sequence for rSREBP-1 is aligned with human
SREBP-1 (hSREBP-1) and rat ADD-1
(rADD-1). The differences in predicted amino acid sequence
are denoted by bold letters. The asterisk
represents the termination codon. A-U-rich regions are
boxed. Numbering for the nucleotides and amino acid
positions are based upon the sequences previously reported in
GenBankTM for hSREBP-1 (accession number P36956) and for
rADD-1 protein sequence (accession number P56720) and for
rADD-1 nucleotide sequence (L16995).
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DISCUSSION |
Supplementing a high carbohydrate diet with oils rich in
(n-6) and (n-3) PUFA results in a rapid
(i.e. <3 h) and coordinate inhibition of hepatic gene
transcription for a wide array of lipogenic enzymes including fatty
acid synthase, acetyl-CoA carboxylase, citrate lyase, malic enzyme, and
stearoyl-CoA desaturase (8, 11, 23, 30). Dose-response studies indicate
that lipogenic gene transcription is suppressed 50% when the diet
contains as little as 3-5% of its energy as PUFA (31). The coordinate
regulation of gene transcription by PUFA suggested that PUFA may employ
a "master switch" type mechanism. The hepatic nuclear factor
SREBP-1 may serve this function (11-14). PUFA appear to reduce the
nuclear content of SREBP-1 by interfering with the proteolytic release of mature SREBP-1 from its endoplasmic reticulum anchored precursor. In
addition, PUFA reduces the amount SREBP-1 precursor by decreasing the
abundance of SREBP-1 mRNA (11-14). In vivo
and cell culture studies indicate that insulin and glucose increase the
hepatic content of SREBP-1 by inducing the SREBP-1 gene
transcription (17, 32). In contrast, glucagon and cAMP suppress
SREBP-1 gene transcription, and consequently block the rise
in hepatic SREBP-1 mRNA and protein elicited by glucose
and insulin (20). Like glucagon and cAMP, treating hepatocytes with
20:4(n-6) also blocked the insulin-glucose induction of
SREBP-1 expression (Fig. 1). However, unlike glucagon and
cAMP, PUFA govern SREBP-1 expression by post-transcriptional
mechanisms (11). Such mechanisms may involve interference with mRNA
processing (33) and/or acceleration of mRNA decay (34). In the case
of SREBP-1, PUFA reduce the hepatic content of
SREBP-1 mRNA by accelerating the rate of mRNA decay.
The liver expresses two forms of SREBP-1, 1a and 1c. SREBP-1 antibody
recognizes both proteins. Consequently, the relative abundance of the
two proteins cannot be quantified. However, due to differences in
splicing, the 5'-end of the SREBP-1c transcript is shorter
than the SREBP-1a mRNA (29). This difference in size permits the use of a ribonuclease protection assay to examine the
influence of PUFA on SREBP-1a and -1c mRNA
abundance and decay. Interestingly, this assay revealed that the
SREBP-1c transcript was more sensitive to PUFA regulation
than was SREBP-1a. Specifically, PUFA reduced the half-life
of SREBP-1c mRNA by 55% while it decreased the
half-life of SREBP-1a by only 35%. Because the decay of
SREBP-1c mRNA was more sensitive to 20:4(n-6)
and 20:5(n-3) feedback, the ratio of
SREBP-1c/1a in the hepatocytes treated with PUFA
decreased from 3:1 to <1:1. The selective loss of SREBP-1c
in response to PUFA is reminiscent of what has been observed in
streptozotocin-diabetic rats (17, 32), but unlike glucagon and insulin
which appear to regulate SREBP-1 gene transcription, PUFA
exert their effects at the level of SREBP-1 mRNA decay.
The mechanisms by which PUFA enhance SREBP-1 decay remain to
be established. Often times the rate-limiting step in mRNA decay is
the shortening of the poly(A) tail (35). The rate of poly(A) tail
shortening appears to depend upon the binding of specific proteins to
"destabilizing" elements within the mRNA (35). One common
destabilizing element is the A-U rich sequences, notably UUAUUUA(U/A)(U/A), located within the 3'-untranslated region of a
transcript (35). Such an A-U-rich region was found to be present within
the 3'-untranslated region of the rat SREBP-1 transcript (Fig. 7), but its functional significance in the decay of
SREBP-1 remains to determined. Moreover, many transcripts
contain A-U-rich instability elements in their 3'-untranslated regions,
but PUFA control of mRNA decay is not a widespread phenomenon.
Thus, the control of SREBP-1 decay by PUFA would appear to
involve more than simply the A-U-rich regions. One alternative
possibility is that the SREBP-1 transcript contains a
destablizing element(s) in the 5'-untranslated region or within
the open reading frame that is targeted by PUFA control mechanisms. In
this respect it is interesting to note that cycloheximide not only
blocked the ability of PUFA to accelerate SREBP-1 decay, but
actually increased the hepatocyte content of both SREBP-1c
and -1a. These results suggest that SREBP-1
mRNA translation is required for mRNA decay, and/or that PUFA
may modulate the activity of a rapidly turning over protein involved in
SREBP-1 mRNA decay. One additional dilemma is why do the
SREBP-1c and -1a transcripts differ in their
response to PUFA-regulated decay. Presumably, SREBP-1c and
-1a both contain the same 3'-untranslated region, but under
the influence of PUFA, the rate of SREBP-1c mRNA decay
is faster than is the decay of SREBP-1a. A key difference
between the two transcripts is that they are produced by two different
promoter sites. This results in the 5'-untranslated region of
SREBP-1c being 45 nucleotides shorter than
SREBP-1a. Thus, one possible explanation for the difference
in decay rates in response to PUFA is that the 5'-untranslated region
of SREBP-1c and -1a plays a regulatory role in
the PUFA-mediated decay of SREBP-1.
In conclusion, dietary (n-6) and (n-3) PUFA have
long been known to decrease the expression of hepatic lipogenic as well
glycolytic genes (8). While recent data indicate that
18:2(n-6) and 18:3(n-3) must undergo desaturation
by the
-6 desaturase to exert their inhibitory effects (36),
numerous studies have failed to link the PUFA control of gene
expression to the production of eicosanoids (31). Moreover, PUFA
exercise their effects in an insulin- and glucagon-independent manner
(37). Despite our inadequate understanding of the signaling mechanisms
employed by PUFA, SREBP-1 is emerging as a transcription factor that is
pivotal to the overall understanding of PUFA regulation of hepatic
lipogenic gene expression. In this story, PUFA exert two effects.
First, PUFA inhibit the proteolytic release of SREBP-1c from its
endoplasmic reticulum anchored precursor which in turn results in an
immediate suppression of lipogenic gene transcription (11, 14). Second,
PUFA accelerate the decay of SREBP-1c mRNA which in turn
lowers the hepatic content of SREBP-1 mRNA and the
synthesis of SREBP-1 precursor. The outcome is a lower capacity for
hepatic lipogenesis, and a decrease in hepatic triglyceride output
(11-13). This ability of PUFA to suppress SREBP-1 expression and thereby exert a strong anti-lipogenic influence not only
explains how PUFA function as hypolipidemic agents but may also offer a
partial explanation for how PUFA improve glucose metabolism and insulin
sensitivity (2-5, 38).