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J Biol Chem, Vol. 273, Issue 52, 35299-35306, December 25, 1998
From the Department of Molecular Genetics, University of Texas,
Southwestern Medical Center, Dallas, Texas 75235
Previous studies have shown that the rate of
fatty acid synthesis is elevated by more than 20-fold in livers of
transgenic mice that express truncated nuclear forms of sterol
regulatory element-binding proteins (SREBPs). This was explained in
part by an increase in the levels of mRNA for the two major enzymes of fatty acid synthesis, acetyl-CoA carboxylase and fatty acid synthase, whose transcription is stimulated by SREBPs. Fatty acid synthesis also requires a source of acetyl-CoA and NADPH. In the current studies we show that the levels of mRNA for ATP citrate lyase, the enzyme that produces acetyl-CoA, are also elevated in the
transgenic livers. In addition, we found marked elevations in the
mRNAs for malic enzyme, glucose-6-phosphate dehydrogenase, and
6-phosphogluconate dehydrogenase, all of which produce NADPH. Finally,
we found that overexpressing two of the SREBPs (1a and 2) led to
elevated mRNAs for stearoyl-CoA desaturase 1 (SCD1), an isoform
that is detectable in nontransgenic livers, and SCD2, an isoform that
is not detected in nontransgenic livers. This stimulation led to an
increase in total SCD activity in liver microsomes. Together, all of
these changes would be expected to lead to a marked increase in the
concentration of monounsaturated fatty acids in the transgenic livers,
and this was confirmed chromatographically. We conclude that expression
of nuclear SREBPs is capable of activating the entire coordinated
program of unsaturated fatty acid biosynthesis in mouse liver.
Sterol regulatory element-binding proteins
(SREBPs)1 are a family of
transcription factors that regulate the low density lipoprotein (LDL)
receptor and multiple enzymes required for the biosynthesis of
cholesterol and fatty acids (see Ref. 1 for review). SREBPs belong to
the basic helix-loop-helix leucine zipper family of transcription
factors. Unlike other members of the basic helix-loop-helix leucine-Zip
family, SREBPs are synthesized as ~1150 amino acid precursors bound
to the endoplasmic reticulum and nuclear envelope. The membrane-bound
precursor must undergo a sequential two-step cleavage process to
release the transcriptionally active NH2-terminal portion
of the protein (2). Once cleaved, the ~500 amino acid NH2-terminal nuclear form enters the nucleus and activates
transcription by binding to promoter regions of genes containing
non-palindromic sterol regulatory elements as well as palindromic
sequences called E-boxes (1, 3, 4). Genes involved in cholesterol
metabolism that are directly activated by SREBPs include the LDL
receptor, 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) synthase, HMG-CoA
reductase, farnesyl diphosphate synthase, squalene synthase, and
SREBP-2 (1, 5-8). Genes involved in fatty acid and triglyceride
synthesis, including acetyl-CoA carboxylase (ACC), fatty acid synthase
(FAS), and glycerol-3-phosphate acyltransferase (GPAT), are also
directly activated by SREBPs (9-11).
To date, three isoforms of SREBP have been identified and characterized
(1). The human versions of all three isoforms were originally
discovered as transcription factors that bound non-palindromic sterol
regulatory elements and regulated cholesterol metabolism. Two of these,
designated SREBP-1a, and -1c, are derived from the same gene through
use of alternative transcription start sites resulting in proteins that
produce different first exons (12). The rat homologue of human SREBP-1c
(adipocyte determination differentiation-dependent factor
1) was originally cloned as a transcription factor that bound to E-box
sequences and promoted adipocyte differentiation (13). The third
isoform, SREBP-2, is derived from a second gene (14). All actively
growing cultured cells studied to date produce predominantly the
SREBP-1a and SREBP-2 isoforms (15). However, in most organs from adult
animals, SREBP-1c and SREBP-2 are predominant (15).
When sterols build up in cells, the proteolytic release of SREBPs from
membranes is inhibited and transcription of all target genes declines
(1). To gain insight into separate roles of each SREBP isoform in
vivo, we previously produced and characterized transgenic mice
that overexpress truncated, transcriptionally active nuclear forms of
human SREBP-1a, -1c, or -2 in liver (16-18). The transgenes encode
versions of the proteins that terminate prior to the membrane
attachment domain. Consequently, these proteins enter the nucleus
directly without a requirement for proteolysis. Mice that overexpressed
the truncated SREBP-1a isoform (TgSREBP-1a) had dramatically enlarged
livers that were engorged with cholesterol as well as triglycerides.
The rates of hepatic cholesterol and fatty acid synthesis were
increased by 5- and 25-fold, respectively, as measured by the
incorporation of [3H]water. Corresponding 5-fold
increases were measured in the liver mRNAs for HMG-CoA synthase,
squalene synthase, and the LDL receptor while 20-25-fold increases
were measured in ACC and FAS (16). In contrast, mice that overexpressed
the nuclear form of human SREBP-1c (TgSREBP-1c) had no increase in
hepatic cholesterol synthesis, but had a 4-fold increase in fatty acid
synthesis. In agreement with the synthesis studies, no increases were
measured in mRNAs for cholesterol synthetic enzymes, but 2- and
4-fold increases were measured in ACC and FAS, respectively (17).
SREBP-2 transgenic mice (TgSREBP-2) had elevated hepatic cholesterol
accumulations like those measured in livers from TgSREBP-1a mice, but
significantly less triglyceride accumulation. The livers from TgSREBP-2
mice had 28-fold higher rates of cholesterol synthesis, but only 4-fold
higher rates of fatty acid synthesis. The mRNAs for the cholesterol
synthetic enzymes, HMG-CoA synthase, farnesyl diphosphate synthase, and
squalene synthase were increased by 10-12-fold. The mRNAs for the
fatty acid synthetic enzymes ACC and FAS were increased 7- and 15-fold,
respectively. Therefore, the SREBP-1 isoforms seem more selective in
activating genes involved in fatty acid synthesis while SREBP-2 is more
specific for activating genes involved in cholesterol synthesis (18).
The mechanism for the relative specificity of each transcription factor
is currently not known.
All three lines of transgenic mice had 3-4-fold increases in the
hepatic mRNA for the fatty acid modifying enzyme, stearoyl-CoA desaturase (SCD). SCD is a microsomal enzyme that introduces a cis-double bond in the Several crucial questions regarding the role of SREBPs in fatty acid
metabolism remain unanswered: 1) SREBPs markedly enhance the
utilization of acetyl-CoA for lipid synthesis, but do they activate
transcription of genes encoding enzymes that synthesize acetyl-CoA? 2)
Do SREBPs increase the mRNAs encoding enzymes that supply the other
substrate for fatty acid synthesis, i.e. NADPH? 3) Which
isoform of SCD is elevated by SREBP overexpression? 4) Does the
contribution of fatty acid overproduction and SCD enhancement lead to a
predominance of unsaturated fatty acids in the transgenic livers? 5)
Are there relative differences among the SREBP isoforms in eliciting
these effects? The current studies were designed to answer these questions.
Materials--
All restriction enzymes were obtained from New
England Biolabs (Beverly, MA) and DNA manipulations were performed
using standard molecular biology techniques (22). Redivue
[ Transgenic Mice--
Transgene constructs encoding amino acids
1-460 of human SREBP-1a, 1-436 of human SREBP-1c, and 1-468 of human
SREBP-2 under the control of the rat phosphoenolpyruvate carboxykinase
promoter were described previously (16-18). Two independent
experiments were designed to study wild-type and SREBP transgenic mice.
In Experiment A, 6 male TgSREBP-1a (line B) (16) and 6 male
nontransgenic wild-type littermate mice were studied. Experiment B
included 5 male TgSREBP-1c (17), 5 male TgSREBP-2 (line B) (18) mice, and 5 male wild-type control mice. The TgSREBP-2 mice studied were
offspring of mice homozygous for the SREBP-2 transgene. Therefore, all
of the wild-type mice in Experiment B were derived from TgSREBP-1c nontransgenic littermates.
All mice were housed in colony cages in a 14-h light/10-h dark cycle
and were maintained on Teklad 6% (w/w) Mouse/Rat Diet 7002 from Harlan
Teklad Premier Laboratory Diets (Madison, WI). Four weeks prior to
sacrifice, transgenic and littermate controls were placed on a low
carbohydrate/high protein diet (number 5789C-3) from Purina Mills Inc.
(St. Louis, MO) containing 71% (w/w) casein and 4.25% (w/w) sucrose.
This diet induces the phosphoenolpyruvate carboxykinase promoter
resulting in maximal transgene expression. All mice were sacrificed at
34-36 weeks of age, during the early phase of the light cycle, and in
the non-fasted state.
Blot Hybridization of RNA--
Total RNA was prepared from mouse
liver using RNeasyTM RNA kit (Qiagen, Chatsworth, CA). For
Northern gel analysis, equal aliquots of total RNA were pooled (total,
15 µg), denatured with formaldehyde and formamide, subjected to
electrophoresis in a 1% formaldehyde agarose gel, and transferred to
Hybond N+ membranes (Amersham) for hybridization. cDNA
probes for ATP citrate lyase, malic enzyme, glucose-6-phosphate
dehydrogenase (Glu-6-PD), 6-phosphogluconate dehydrogenase (PGDH),
glycerol-3-phosphate acyltransferase (GPAT), and S14 were prepared by
reverse transcriptase-polymerase chain reaction using mouse liver
poly(A)+ RNA as a template as described previously (16).
The polymerase chain reaction primers used to generate these probes are
as follows. ATP citrate lyase: 5',5'-AAGTAGACACTGGTCTGTAACATGGAACAT-3'
and 3',5'-TCCAGAACACATGAGCATGTAACTGAGCCA-3' (23, 24). Malic enzyme: 5',5'-AATTAAGAATTTCGAACGACTGAACTCTGA-3' and
3',5'-TCGTTAAATGTGCAATACTTGTTTCGATAC-3' (25). Glu-6-PD:
5',5'-CCAGGTGTGTGGGATCCTGAGGGAAGAGTT-3' and 3',5'-GGTCCAAAGATCCTGTTGGCAAACCTCAGC-3' (26). PGDH:
5',5'-TGGT(G/C)TCCAAGCTGAAGAAGCC-3' and 3',5'-TCTTTGGCAGCAGGTGTTTGCC-3'
(27, GenBank accession number U30255), GPAT:
5',5'-TCCAGAAACCCAGCAGACGA-3' and 3',5'-CAGCACCACAAAACTCAGAA-3' (11).
S14: 5',5'-ATGCAAGTGCTAACGAAACGC-3' and
3',5'-AGAAGTGCAGGTGGAACTGGGC-3' (28). Northern blot analysis was
carried out as described previously (16). The bands detected by
Northern analysis were quantified by exposing the filter to a BAS1000
Fuji PhosphorImager (Tokyo, Japan), and the results were normalized to
the signal generated by glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) mRNA.
RNase Protection Assay--
cRNA probes and protection assay
conditions for comparison of transgene expression in livers of
SREBP-1a, -1c, and -2 mice were prepared as described previously (18).
cDNA fragments for mouse stearoyl-CoA desaturase 1 (SCD1) and
stearoyl-CoA desaturase 2 (SCD2) were amplified by polymerase chain
reaction from first strand cDNA prepared from mouse epididymal fat
pad poly(A)+ RNA using the following primers. SCD1:
5',5'-CCGGCCCACATGCTCCAAGAGATCTCCAGT-3' and
3',5'-GTACTCCAGCTTGGGCGGGGGTCCCTCCTC-3' (20). SCD2:
5',5'-CTGCAAGAGATCTCTGGCGCTTACTCAGCC-3' and
3',5'-CTCATCATCCTGATAGGTGGGGTCATATAG-3' (21). The RNase protection assay was carried out as described previously except that
the amount of cRNA probes for SCD1 and SCD2 was increased to 4 × 105 cpm (15). The specific activities of the cRNAs were
0.9-1.4 × 109 cpm/µg for SCD1 and SCD2 and
1.1-1.6 × 109 cpm/µg for Stearoyl-CoA Desaturase Activity--
Stearoyl-CoA desaturase
activity was measured in liver microsomes that were prepared by
homogenizing ~100 mg of liver in 0.5 ml of a 0.1 M
potassium phosphate buffer (pH 7.4). The homogenate was centrifuged at
10,000 × g at 4 °C for 30 min and the resultant supernatant was then centrifuged at 100,000 × g at
4 °C for 30 min. The microsomal pellet was resuspended in a 0.1 M potassium phosphate buffer (pH 7.2) (29). Protein
concentrations were determined by the method of Lowry (30). The
reaction mixture for the stearoyl-CoA desaturase activity included the
following: 60 µM [1-14C]stearoyl-CoA, 2 mM NADH in 10 mM potassium phosphate (pH 7.2), 0.1 M potassium phosphate (pH 7.2), and 100 µg of
microsomal protein in a final volume of 100 µl (31, 32). The reaction
conditions, saponification, and fatty acid extractions were carried out
as described (32). [3H]Oleic acid (~2000 dpm/µl),
oleic acid (25 µg/µl), and stearic acid (25 µg/µl) were added
as recovery markers and the extracts were evaporated to dryness under
air. Methyl esters were then prepared as described and evaporated to
dryness (32).
The products were resuspended in 30 µl of chloroform and spotted on
5% activated argentation plates to achieve separation by thin layer
chromatography (TLC) (33). The TLC plates were run in 100% benzene,
allowed to dry and sprayed with 0.05% Rhodamine B in methanol. The
resulting bands were visualized under UV light and the appropriate
fractions were cut into scintillation vials (34). Radioactivity was
determined using a liquid scintillation spectrometer. The enzyme
activity is expressed as nanomole·min Tissue Cholesterol and Fatty Acid Composition--
Total lipids
were extracted from 300-350-mg aliquots of liver and esterified
cholesterol, triglycerides, and total phospholipids were separated on
500-mg silica columns (Varian catalog number 1211-3036) exactly as
described (35). After saponification, the fatty acids in each of these
samples were methyl esterified and the relative abundance of each fatty
acid was quantified by gas-liquid chromatography (36, 37). Liver
unesterified and esterified cholesterol also were quantified by GLC
(35, 38).
Table I shows the phenotypic
characteristics of the mice used in the current studies. Experiment A
included mice overexpressing the nuclear form of human SREBP-1a and
their wild-type littermates, while Experiment B included mice that
overexpress the nuclear form of human SREBP-1c or SREBP-2. Wild-type
littermates from the TgSREBP-1c mice were used as controls in
Experiment B. The changes in plasma and liver lipid levels
between wild-type and transgenic mice are consistent with previously
published results (16-18). Of note, TgSREBP-1a mice had mean liver
cholesteryl ester and triglyceride concentrations of 9.9 and 183 mg/g,
respectively. Both levels were ~30-fold higher than those measured in
control livers. SREBP-1c overexpression resulted in very little change in the cholesteryl ester concentration but it did increase the triglyceride concentration to 36 mg/g. TgSREBP-2 mouse livers had an
elevated mean cholesteryl ester concentration of 3.1 mg/g and a
triglyceride content of 38 mg/g.
Fig. 1 compares the levels of transgene
expression in the livers of the mice described in Table I as measured
by the RNase protection assay. In order to estimate the relative
expression of each transgene, we designed a cRNA probe that contained
the same number of 32P atoms in the protected fragment. The
human SREBP cRNA probes did not produce a protected band in wild-type
mouse liver. The level of mRNA encoding the SREBP-1a transgene was
arbitrarily set at 1. The SREBP-1c and SREBP-2 transgenes were
expressed at levels that were 40 and 60% of the levels of SREBP-1a.
The difference in transgene expression precludes any direct comparisons
between the three lines of transgenic mice regarding the absolute
effects of each SREBP isoform, but they do permit an analysis of the
relative ability of each isoform to affect one mRNA
versus another.
Nuclear Sterol Regulatory Element-binding Proteins Activate Genes
Responsible for the Entire Program of Unsaturated Fatty Acid
Biosynthesis in Transgenic Mouse Liver*
,
ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References
9 position of saturated fatty
acids, resulting in the production of monounsaturated fatty acids (19).
The preferred substrates for SCD are palmitoyl (16:0) and stearoyl
(18:0) CoA which are converted to palmitoleoyl (16:1) and oleoyl (18:1)
CoA, respectively. Two isoforms of SCD, SCD1 and SCD2, are currently
known (20, 21). Most organs produce both SCD1 and SCD2. The notable
exception is liver, which expresses only the SCD1 isoform (21). The
regulation of SCD is of considerable physiologic importance since this
enzyme ultimately determines the ratio of monounsaturated to saturated fatty acids in the cell. Alterations in this ratio can change membrane
fluidity. Such alterations have been implicated in various disease
processes, including cancer, diabetes, obesity, and vascular disease
(19).
EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References
-32P]dCTP (3000 Ci/mmol) was obtained from Amersham
Corp. Oleic acid [9,10-3H]- and
[1-14C]stearoyl-CoA were purchased from American
Radiolabeled Chemicals, Inc. (St. Louis, MO). Stearic acid, oleic acid,
and silica gel plates were purchased from Alltech (Deerfield, IL),
stearoyl-CoA from Fluka (Ronkonkoma, NY), and silver nitrate from
Calbiochem (La Jolla, CA). All other chemicals used were from Sigma.
The content of cholesterol and triglycerides in plasma and liver was measured as described previously (16).
-actin.
1·mg
protein1.
RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References
Phenotypic comparison of wild-type and transgenic mice
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[in a new window]
Fig. 1.
Relative amounts of hepatic mRNAs
encoding the truncated human transgene as measured by the RNase
protection assay. Total RNA from the livers of wild-type
(lanes A and B), TgSREBP-1a (lane C),
TgSREBP-1c (lane D), and TgSREBP-2 (lane E) mice
was isolated from the mice described in Table I. Equal aliquots of
total RNA (10 µg) were hybridized at 68 °C to a
32P-labeled cRNA probe for human SREBP-1 (lanes A,
C, and D) or SREBP-2 (lanes B and
E) to detect the transgene product. Protected fragments were
separated by gel electrophoresis and exposed to film at 
80 °C with
an intensifying screen. The radioactivity in the gel was quantified by
exposure of the filters to a Bio-Imaging Analyzer with BAS1000 MacBas
software (Fuji Medical Systems), normalized to the signal generated by
-actin, and expressed as the fold change relative to the value of
the mRNA encoding the SREBP-1a transgene.
Northern blot experiments were used to further examine the effects of SREBP overexpression on hepatic genes involved in fatty acid metabolism (Fig. 2). ATP citrate lyase generates acetyl-CoA, which is the substrate for cholesterol and fatty acid synthesis. ATP citrate lyase mRNA was increased 4.4-, 3.2-, and 1.5-fold in livers of TgSREBP-1a, -1c, and -2 mice, respectively. The fold changes in the mRNAs for the fatty acid synthetic enzymes, ACC and FAS, were slightly less than previously reported (18), most likely because the mice were sacrificed in the fed state. GPAT, which catalyzes the first committed step in triglyceride synthesis, was significantly increased in livers from SREBP-1a and -1c mice, but not in the SREBP-2 animals. The measured increase was greatest in the TgSREBP-1a mice.
Malic enzyme, Glu-6-PD, and PGDH are enzymes involved in the generation of NADPH required for fatty acid synthesis. The mRNAs for these genes are shown in the second row of Fig. 2. SREBP-1a overexpression increased the hepatic mRNAs for malic enzyme and PGDH by 11- and 6.6-fold, respectively. The most dramatic change was a 31-fold increase in the Glu-6-PD mRNA. SREBP-1c and SREBP-2 overexpression resulted in significant but smaller increases in these mRNAs. S14, a gene known to be induced during lipogenesis (39), was also significantly increased in livers from TgSREBP-1a and -1c mice. However, in livers from TgSREBP-2 mice the mRNA for S14 was slightly less than that measured in control livers. To date, S14 and GPAT are the only genes that have been found to be selectively induced only by the SREBP-1 isoforms in liver. The fold increase measured in the mRNA for the cholesterol synthetic enzyme, HMG-CoA reductase, was greatest in the TgSREBP-2 mice. All fold changes reported in hepatic mRNAs were calculated after correction for loading differences using the signal generated by GAPDH. GAPDH is a glycolytic enzyme, and as such, could be increased in SREBP transgenic mice owing to the apparent increase in lipogenesis. Therefore, the fold increases reported for the various hepatic mRNAs may be slightly underestimated.
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Stearoyl-CoA desaturase is the key enzyme responsible for the synthesis of monounsaturated fatty acids (19). We have previously reported that the mRNA for SCD is increased in livers from all three lines of transgenic mice as measured by Northern blotting (16-18). Although SCD1 is reportedly the only SCD isoform present in liver (21), the cDNA probe for SCD used in our previous Northern blots could cross-hybridize with SCD2, if present. To determine whether the measured increase in SCD mRNA in livers from SREBP transgenic mice was due to an increase in SCD1 and/or SCD2 mRNA, we designed cRNA probes for use in a RNase protection assay that would specifically detect SCD1 or SCD2 mRNA.
In order to be certain that the level was within the linear range of the RNase protection assay, we added various amounts of mRNA to the assay tubes. Fig. 3 shows that the intensities of the protected fragments of the SCD1 mRNA increased as increasing amounts of RNA were added to the incubation. Quantifying the protected fragments verified the assay was linear up to 10 µg of total RNA from all lines of transgenic mice.
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Fig. 4 shows the results of the RNase protection assay for SCD1 and SCD2 mRNA transcripts in livers from the wild-type and transgenic mice described in Table I. Lanes 1 and 2 show the protected bands for SCD1 and SCD2 in 5 µg of total RNA from the epididymal fat pads of wild-type mice. SCD1 and SCD2 transcripts are both known to be present in white adipose tissue, and therefore, this RNA was used as a positive control (20, 21). Lanes 3-7 show the protected fragments for SCD1 in livers of the wild-type and transgenic mice described in Table I. The SCD1 mRNA was increased by 2-fold in all three lines of transgenic mice. Lanes 8-12 show the protected mRNA fragments for SCD2. No SCD2 transcript could be detected in livers from wild-type or TgSREBP-1c mice. However, a protected band for SCD2 was detected in RNA from livers of TgSREBP-1a and TgSREBP-2 mice (lanes 9 and 12). The amount of SCD2 transcript in TgSREBP-2 livers is equivalent to the amount found in white fat from wild-type mice, whereas TgSREBP-1a livers had 7-fold more SCD2 transcript than that measured in white fat.
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To determine whether the measured increase in SCD mRNA from the transgenic mice resulted in increased enzyme activity, we measured SCD activity in liver microsomes from wild-type and transgenic mice. Table II shows the mean SCD activities as estimated by the rate of conversion of [1-14C]stearoyl-CoA to [1-14C]oleate by hepatic microsomes. TgSREBP-1a mouse liver microsomes had 5-fold higher SCD enzymatic activities than microsomes from littermate wild-type mice. The activities in livers from TgSREBP-1c and TgSREBP-2 mice were both approximately 2.5-fold higher than wild-type control levels.
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The major substrates for SCD are palmitic (16:0) and stearic (18:0) acids, which are converted to palmitoleic (16:1) and oleic (18:1) acids, respectively (19). To determine whether the transgenic mouse livers contained more monounsaturated fatty acids, the various lipid fractions were separated and the relative fatty acid composition in each fraction was measured. Table III shows the relative percentage of 8 major fatty acids measured in the various lipid fractions from wild-type and transgenic mouse livers. In total lipid extracts, TgSREBP-1a livers had a 3-fold increase in the relative amount of palmitoleic acid (16:1) and a similar 3.6-fold increase in the relative amount of oleic acid (18:1). On the other hand, the relative amounts of palmitic (16:0) and stearic (18:0) acids were decreased. The total lipid extracts from livers of TgSREBP-1c and TgSREBP-2 mice had similar 3-fold increases in the relative amount of palmitoleic acid and 2-fold increases in the amount of oleic acid.
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The hepatic cholesteryl ester fractions from all three lines of transgenic mice contained a 2-fold increase in the relative amount of palmitoleic acid (16:1), while the relative amount of oleic acid (18:1) was 4.8-fold higher in TgSREBP-1a mice and 2.5-fold higher in TgSREBP-1c and -2 mice (Table III). The changes measured in the relative amounts of monounsaturated and saturated fatty acids in the triglyceride fractions from livers of the TgSREBP-1a, -1c, and -2 mice are similar to the changes measured in the cholesteryl esters. Inasmuch as the cholesteryl ester and triglyceride content in livers of TgSREBP-1a and TgSREBP-2 mice are significantly elevated (see Table I), the absolute monounsaturated fatty acid content in each fraction is dramatically increased. Fatty acid compositional changes in the phospholipid fractions were much less striking, although small increases in the percentage of oleic acid were measured in livers from all lines of transgenic mice (Table III).
The relative increase in the percentage of monounsaturated fatty acids (16:1 and 18:1) measured in livers of transgenic mice was accompanied by significant reductions in the relative percentage of saturated fatty acids (16:0 and 18:0). Fig. 5 shows the ratio of monounsaturated to saturated fatty acids measured in the livers from wild-type and the three lines of transgenic mice. In total lipid extracts (panels A and D), the 16:1/16:0 ratio was increased in the livers from TgSREBP-1a and TgSREBP-2 mice, but not in livers from TgSREBP-1c mice. The 18:1/18:0 ratio was significantly elevated only in livers from TgSREBP-1a mice. In the cholesteryl ester fractions (panels B and E) and triglyceride fractions (panels C and F), the ratio of monounsaturated to saturated fatty acids was significantly increased in all three lines of transgenic mice. The most pronounced changes were again present in livers from TgSREBP-1a mice. The 16:1/16:0 ratios in the cholesteryl ester fractions from TgSREBP-1a, -1c, and -2 livers were increased by 10-, 3-, and 6-fold, respectively (panel B), while the 18:1/18:0 ratios were increased by 23-, 9-, and 10-fold, respectively (panel E). The hepatic triglyceride fractions also contained increases in the 16:1/16:0 and 18:1/18:0 ratios (panels C and F), although the absolute fold-change is approximately one-third of that measured in the cholesteryl ester fractions.
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DISCUSSION |
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Top
Abstract
Introduction
Procedures
Results
Discussion
References
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The purpose of the current study was to gain insight into the effects of SREBP overexpression on selected aspects of fatty acid metabolism in liver using transgenic mice. In previous studies, we observed increased rates of hepatic fatty acid synthesis in all three lines of transgenic mice as measured by the incorporation of [3H]water into newly synthesized fatty acids (16, 18). The genes required to produce the substrates for fatty acid synthesis as well as the final fatty acid products have not been studied. Inasmuch as TgSREBP-1a mouse livers consistently showed the greatest changes in fatty acid metabolism, the initial discussion will focus on the results obtained in these mice.
Most of the carbon source for fatty acid synthesis comes from pyruvate, whose carbons are incorporated into citrate within mitochondria (40). The citrate then leaves the mitochondria and enters the cytoplasm where ATP citrate lyase cleaves off a two-carbon unit as acetyl-CoA, which is then incorporated into fatty acids and cholesterol. SREBP-1a overexpression resulted in a 4-fold increase in the mRNA for ATP citrate lyase, which would thereby increase the acetyl-CoA substrate required for lipid synthesis.
The synthesis of fatty acids requires a second substrate, NADPH, which
supplies hydrogen atoms (40). NADPH is also required for the
desaturation reaction catalyzed by SCD (19). Two major pathways exist
for the generation of NAPDH, and each is estimated to contribute
approximately 50% in rat liver (41). The first reaction is the
oxidative decarboxylation of malate to form pyruvate and
CO2. This reaction is catalyzed by malic enzyme, which
simultaneously generates NADPH from NADP. The second
source of NADPH is from the pentose phosphate shunt. Glu-6-PD and PGDH
are the first two enzymes in this pathway and both generate NADPH (40).
The livers of SREBP-1a transgenic mice had significantly elevated
mRNAs for all three NADPH producing enzymes, indicating both
pathways for NAPDH synthesis were activated. Therefore, SREBP-1a not
only activates the genes directly responsible for fatty acid synthesis,
ACC and FAS, but it also activates the genes responsible for the
production of the acetyl-CoA and NADPH required for fatty acid
synthesis. Since lipogenesis is globally stimulated in TgSREBP-1a
livers, it is possible that transcription of the genes encoding the
enzymes responsible for the production of citrate are also increased
(i.e. glucokinase, phosphofructokinase I, and pyruvate
kinase), although these mRNAs were not studied in the current experiments.
To date, SREBP-1a has been shown to directly bind to the promoters and activate the transcription of ACC, FAS, and GPAT (9-11). To our knowledge, the promoters for ATP citrate lyase, malic enzyme, Glu-6-PD, and PGDH have not been studied. Therefore, we cannot conclude SREBP-1a activates the transcription of these genes directly. However, whether the effect is direct or indirect, the end result is an increase in mRNAs encoding multiple enzymes required for the simultaneous production of cholesterol, fatty acids, and triglycerides.
The transcriptional activation of SCD2 by SREBP-1a and SREBP-2 in liver was unexpected. This mRNA was reported to be absent from mouse liver (21) and indeed we failed to detect it in livers of wild-type mice. It did appear in livers of mice expressing SREBP-1a and SREBP-2, indicating that the block in SCD2 expression in liver is not complete and that it might be expressed if the stimulus is strong enough.
The combined increase in SCD1 and SCD2 mRNAs led to an increase in the measured SCD activity in liver microsomes (Table II). This is of possible metabolic importance because the predominant SCD product, oleic acid, is the preferred substrate for acyl-CoA:cholesterol acyltransferase, the enzyme responsible for the esterification of cholesterol (42). The increased esterification of cholesterol prevents the accumulation of potentially toxic-free cholesterol in TgSREBP-1a mouse livers and increases the availability of esterified cholesterol for export in the form of VLDL.2 As predicted, the increase in SCD activity resulted in a significant increase in the monounsaturated fatty acid content of cholesteryl esters. A very similar fatty acid profile was measured in the triglyceride fractions. Approximately 80% of the fatty acids present in these fractions were monounsaturated with the vast majority being oleic acid. The changes in liver cholesteryl ester and triglyceride fatty acid composition were reflected in the lipids secreted in VLDL from TgSREBP-1a mice.2 The marked hepatic enrichment of oleic acid can potentially have a wide range of effects on lipid metabolism, such as increased VLDL secretion and accelerated atherosclerosis (43, 44). In cultured cells, the addition of oleic acid to the medium is required for maximal VLDL secretion (44). Therefore, it might be predicted the TgSREBP-1a mice have increased rates of VLDL secretion. This hypothesis is currently under investigation.
The changes measured in the mRNAs for fatty acid synthetic enzymes in livers of TgSREBP-1c and TgSREBP-2 mice were in a similar direction to those observed in the TgSREBP-1a animals, but they were of lower magnitude. In part, this may be attributed to the slightly lower expression of the SREBP-1c and -2 transgenes as compared with TgSREBP-1a (0.4- and 0.6-fold, respectively). The mRNAs for ACC, FAS, and the NAPDH producing enzymes were increased 2-4-fold in TgSREBP-1c and TgSREBP-2 livers. These changes are consistent with the 4-fold increases measured in the rates of fatty acid synthesis using [3H]water in both TgSREBP-1c and TgSREBP-2 mouse livers (18). Interestingly, there was a significant difference between the two SREBP-1 isoforms and SREBP-2 in their ability to activate the lipogenic genes GPAT and S14. Neither mRNA was changed in TgSREBP-2 mouse livers, but both were significantly increased in TgSREBP-1c and -1a livers. In the case of GPAT, this finding is consistent with GPAT promoter-reporter studies in which the relative stimulation of promoter activity was 5.5-fold greater with SREBP-1a than SREBP-2 in HepG2 cells (11). This observation is also consistent with the idea that SREBP-1 is relatively selective as compared with SREBP-2 in activating lipogenic genes (18). The molecular basis of such isoform selectivity is under active study.
The hepatic fatty acid compositions in each lipid fraction were remarkably similar in TgSREBP-1c and -2 mice. Livers from both lines of transgenic mice were enriched with oleic acid in the cholesteryl ester and triglyceride fractions, but to levels that were approximately one-half those measured in TgSREBP-1a livers. The increase in SCD activity was also identical in the TgSREBP-1c and TgSREBP-2 livers. Interestingly, TgSREBP-1c livers did not contain mRNA for SCD2. Whether this is due to lower transgene expression, or to the inability of SREBP-1c to transcriptionally activate SCD2 is not known.
The studies in this paper and the previous papers (16-18) on
transgenes encoding truncated SREBPs indicate the potential of high-level expression of nuclear SREBPs to activate the programs for
synthesis of unsaturated fatty acids and cholesterol in liver. However,
they do not tell us whether the SREBPs carry out this activation under
normal physiologic conditions. Answers to this question will come only
when methods are found to block the activities of nuclear SREBPs in
liver or to modify the target genes so they lose their ability to
respond to SREBPs.
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ACKNOWLEDGEMENTS |
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We thank Drs. Michael S. Brown and Joseph L. Goldstein for their continued encouragement and critical reading of the manuscript. We also thank Dr. Stephen Turley and Dr. John Dietschy for access to the GLC and Robin Craddock, Scott Clark, Debra Morgan, and Richard Gibson for excellent technical assistance.
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FOOTNOTES |
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* This work was supported in part by National Institutes of Health Grant HL-20948, the Moss Heart Foundation, and the Perot Family Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a Research Fellowship from the Manpei Suzuki Diabetes
Foundation of Tokyo, Japan.
§ Supported by the Medical Scientist Training Grant GM08014.
¶ To whom correspondence should be addressed: Dept. of Molecular Genetics, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Rm. L5-238, Dallas, TX 75235. Tel.: 214-648-2141; Fax: 214-648-8804.
The abbreviations used are: SREBP, sterol regulatory element-binding protein; ACC, acetyl-CoA carboxylase; FAS, fatty acid synthase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GPAT, glycerol-3-phosphate acyltransferase; Glu-6-PD, glucose-6-phosphate dehydrogenase; HMG-CoA, 3-hydroxy-3-methylglutaryl coenzyme A; VLDL, very low density lipoprotein; PGDH, 6-phosphogluconate dehydrogenase; SCD, stearoyl-CoA desaturase.
2 J. D. Horton, H. Shimano, J. L. Goldstein, and M. S. Brown, unpublished observations.
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