THE JOURNAL OF BIOLOGICAL CHEMISTRY© 1998 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 273, No. 52, Issue of December 25, pp. 35299 –35306, 1998
Printed in U.S.A.

Nuclear Sterol Regulatory Element-binding Proteins Activate Genes
Responsible for the Entire Program of Unsaturated Fatty Acid
Biosynthesis in Transgenic Mouse Liver*(Received for publication, August 3, 1998, and in revised form, September 15, 1998)

Iichiro Shimomura‡, Hitoshi Shimano, Bobby S. Korn§, Yuriy Bashmakov, and Jay D. Horton¶
From the Department of Molecular Genetics, University of Texas, Southwestern Medical Center, Dallas, Texas 75235

Previous studies have shown that the rate of fatty acidsynthesis is elevated by more than 20-fold in livers oftransgenic mice that express truncated nuclear forms ofsterol regulatory element-binding proteins (SREBPs).
This was explained in part by an increase in the levels ofmRNA for the two major enzymes of fatty acid synthesis,
acetyl-CoA carboxylase and fatty acid synthase, whosetranscription 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 mRNAfor ATP citrate lyase, the enzyme that produces acetylCoA, are also elevated in the transgenic livers. In addition, we found marked elevations in the mRNAs formalic enzyme, glucose-6-phosphate dehydrogenase, and6-phosphogluconate dehydrogenase, all of which produce NADPH. Finally, we found that overexpressing twoof the SREBPs (1a and 2) led to elevated mRNAs forstearoyl-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 inliver 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 capableof activating the entire coordinated program of unsaturated fatty acid biosynthesis in mouse liver.

Sterol regulatory element-binding proteins (SREBPs)1 are afamily of transcription factors that regulate the low density
lipoprotein (LDL) receptor and multiple enzymes required forthe biosynthesis of cholesterol and fatty acids (see Ref. 1 forreview). SREBPs belong to the basic helix-loop-helix leucine* 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 partby the payment of page charges. This 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.
1
The abbreviations used are: SREBP, sterol regulatory elementbinding 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.
This paper is available on line at http://www.jbc.org

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-boundprecursor 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 infatty 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 andcharacterized (1). The human versions of all three isoforms
were originally discovered as transcription factors that boundnon-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 alternativetranscription 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 toE-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 ofeach 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 theproteins 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 cholesteroland 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 the35299

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Nuclear SREBPs Activate Unsaturated Fatty Acid Biosynthesis

LDL receptor while 20 –25-fold increases were measured in
ACC and FAS (16). In contrast, mice that overexpressed thenuclear 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 cholesterolsynthetic 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-foldhigher rates of cholesterol synthesis, but only 4-fold higher
rates of fatty acid synthesis. The mRNAs for the cholesterolsynthetic enzymes, HMG-CoA synthase, farnesyl diphosphatesynthase, 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 specificfor 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 D9 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 isliver, which expresses only the SCD1 isoform (21). The regulation of SCD is of considerable physiologic importance since thisenzyme ultimately determines the ratio of monounsaturated tosaturated fatty acids in the cell. Alterations in this ratio canchange membrane fluidity. Such alterations have been implicated in various disease processes, including cancer, diabetes,
obesity, and vascular disease (19).
Several crucial questions regarding the role of SREBPs infatty 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 themRNAs encoding enzymes that supply the other substrate forfatty acid synthesis, i.e. NADPH? 3) Which isoform of SCD iselevated 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.
EXPERIMENTAL PROCEDURES

Materials—All restriction enzymes were obtained from New England
Biolabs (Beverly, MA) and DNA manipulations were performed using
standard molecular biology techniques (22). Redivue [a-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 gelplates 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).
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 carboxyki-

nase 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 mg),denatured with formaldehyde and formamide, subjected to electrophoresis in a 1% formaldehyde agarose gel, and transferred to Hybond
N1 membranes (Amersham) for hybridization. cDNA probes for ATP
citrate lyase, malic enzyme, glucose-6-phosphate dehydrogenase (Glu6-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)1 RNA asa template as described previously (16). The polymerase chain reaction
primers used to generate these probes are as follows. ATP citrate lyase:59,59-AAGTAGACACTGGTCTGTAACATGGAACAT-39 and 39,59-TCCAGAACACATGAGCATGTAACTGAGCCA-39 (23, 24). Malic enzyme:
59,59-AATTAAGAATTTCGAACGACTGAACTCTGA-39 and 39,59-TCGTTAAATGTGCAATACTTGTTTCGATAC-39 (25). Glu-6-PD: 59,59-CCAGGTGTGTGGGATCCTGAGGGAAGAGTT-39 and 39,59-GGTCCAAAGATCCTGTTGGCAAACCTCAGC-39 (26). PGDH: 59,59-TGGT(G/C)TCCAAGCTGAAGAAGCC-39 and 39,59-TCTTTGGCAGCAGGTGTTTGCC-39 (27, GenBank accession number U30255), GPAT: 59,59-TCCAGAAACCCAGCAGACGA-39 and 39,59-CAGCACCACAAAACTCAGAA-39(11). S14: 59,59-ATGCAAGTGCTAACGAAACGC-39 and 39,59-AGAAGTGCAGGTGGAACTGGGC-39 (28). Northern blot analysis was carriedout 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 fromfirst strand cDNA prepared from mouse epididymal fat pad poly(A)1
RNA using the following primers. SCD1: 59,59-CCGGCCCACATGCTCCAAGAGATCTCCAGT-39 and 39,59-GTACTCCAGCTTGGGCGGGGGTCCCTCCTC-39 (20). SCD2: 59,59-CTGCAAGAGATCTCTGGCGCTTACTCAGCC-39 and 39,59-CTCATCATCCTGATAGGTGGGGTCATATAG-39 (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 3 105 cpm (15). The specific activities of the cRNAs
were 0.9 –1.4 3 109 cpm/mg for SCD1 and SCD2 and 1.1–1.6 3 109
cpm/mg for b-actin.
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 (pH7.4). The homogenate was centrifuged at 10,000 3 g at 4 °C for 30 min
and the resultant supernatant was then centrifuged at 100,000 3 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 thestearoyl-CoA desaturase activity included the following: 60 mM[1-14C]stearoyl-CoA, 2 mM NADH in 10 mM potassium phosphate (pH7.2), 0.1 M potassium phosphate (pH 7.2), and 100 mg of microsomal
protein in a final volume of 100 ml (31, 32). The reaction conditions,saponification, and fatty acid extractions were carried out as described(32). [3H]Oleic acid (;2000 dpm/ml), oleic acid (25 mg/ml), and stearicacid (25 mg/ml) were added as recovery markers and the extracts were
evaporated to dryness under air. Methyl esters were then prepared as

Nuclear SREBPs Activate Unsaturated Fatty Acid Biosynthesis

35301

TABLE I
Phenotypic comparison of wild-type and transgenic mice
Each value represents the mean 6 S.E. In Experiment A, TgSREBP-1a mice were derived from line B in Fig. 4 of Ref. 16, and nontransgenic
littermates of 2 litters were used as controls. In Experiment B, TgSREBP-1c mice were derived from mice described in Ref. 17, and TgSREBP-2
mice were derived from line B in Ref. 18. Nontransgenic littermates from 2 litters of TgSREBP-1c were used as controls. All mice in Experiments
A and B were 34 –36 weeks of age, and were fed the low carbohydrate/high protein diet for 4 weeks before sacrifice. Plasma lipids were measured
from nonfasted samples.
Genotype of miceParameter

6 males

Experiment A
Body weight (g)Liver weight (g)Liver cholesterol content (mg/g)
Liver esterified cholesterol content (mg/g)
Liver triglyceride content (mg/g)
Total plasma cholesterol (mg/dl)
Total plasma triglyceride (mg/dl)

Wild-typeTgSREBP-1a29.7 6 0.8
1.44 6 0.06
2.44 6 0.1
0.30 6 0.04
6.22 6 0.8
95 6 5.4
205 6 1932.7 6 1.1
5.89 6 0.33a
12.0 6 1.2a9.9 6 0.89a
183 6 11a40 6 4.6a
77 6 12a

Wild-typeTgSREBP-1c

TgSREBP-2

30.0 6 0.8
1.52 6 0.08
2.28 6 0.1
0.1 6 0.01
5.17 6 0.1
100 6 5.7
158 6 1131.0 6 0.9
1.59 6 0.08
2.96 6 0.1a
0.55 6 0.09b
36.1 6 6.3b
123 6 5.7
108 6 1428.4 6 0.7
1.66 6 0.09
5.76 6 0.3a
3.1 6 0.06a
37.9 6 6.8b
105 6 4.3
74.2 6 7.7a

5 males

Experiment B
Body weight (g)Liver weight (g)Liver cholesterol content (mg/g)
Liver esterified cholesterol content (mg/g)
Liver triglyceride content (mg/g)
Total plasma cholesterol (mg/dl)
Total plasma triglycerides (mg/dl)ab

p , 0.001 between wild-type and transgenic mice (Student’s t test).
p , 0.01 between wild-type and transgenic mice (Student’s t test).

described and evaporated to dryness (32).
The products were resuspended in 30 ml 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 isexpressed as nanomolezmin21zmg protein21.
Tissue Cholesterol and Fatty Acid Composition—Total lipids wereextracted 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 wasquantified by gas-liquid chromatography (36, 37). Liver unesterified
and esterified cholesterol also were quantified by GLC (35, 38).
RESULTS

Table I shows the phenotypic characteristics of the mice usedin the current studies. Experiment A included mice overexpressing the nuclear form of human SREBP-1a and their wildtype 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 ascontrols 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. Bothlevels 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 thetriglyceride 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 thelivers 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 protectedfragment. 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 differencein transgene expression precludes any direct comparisons between the three lines of transgenic mice regarding the absoluteeffects of each SREBP isoform, but they do permit an analysisof the relative ability of each isoform to affect one mRNA versus
another.
Northern blot experiments were used to further examine theeffects of SREBP overexpression on hepatic genes involved infatty acid metabolism (Fig. 2). ATP citrate lyase generates
acetyl-CoA, which is the substrate for cholesterol and fatty acidsynthesis. 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 weresacrificed in the fed state. GPAT, which catalyzes the firstcommitted 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 formalic 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 insignificant 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

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Nuclear SREBPs Activate Unsaturated Fatty Acid Biosynthesis

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

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 mg) 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) todetect the transgene product. Protected fragments were separated bygel electrophoresis and exposed to film at 280 °C with an intensifying
screen. The radioactivity in the gel was quantified by exposure of thefilters to a Bio-Imaging Analyzer with BAS1000 MacBas software (Fuji
Medical Systems), normalized to the signal generated by b-actin, andexpressed as the fold change relative to the value of the mRNA encoding
the SREBP-1a transgene.

be slightly underestimated.
Stearoyl-CoA desaturase is the key enzyme responsible forthe synthesis of monounsaturated fatty acids (19). We havepreviously reported that the mRNA for SCD is increased inlivers 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 increasein 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 theintensities 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 mg of total RNA from all lines of transgenic
mice.
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 mg of total RNA
from the epididymal fat pads of wild-type mice. SCD1 andSCD2 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 describedin Table I. The SCD1 mRNA was increased by 2-fold in all threelines of transgenic mice. Lanes 8 –12 show the protected mRNAfragments for SCD2. No SCD2 transcript could be detected inlivers 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 thatmeasured in white fat.
To determine whether the measured increase in SCD mRNA
from the transgenic mice resulted in increased enzyme activity,

FIG. 2. Amounts of mRNAs of genes involved in fatty acid metabolism from livers of wild-type and transgenic mice as measured
by blot hybridization. The mice used in this experiment are described in Experiments A and B of Table I. Total RNA isolated from livers of mice
was pooled (15 mg), and subjected to electrophoresis and blot hybridization with the indicated 32P-labeled probe. The amount of radioactivity in eachband was quantified as described in Fig. 1. The fold change in each mRNA, relative to that of the wild-type control, was calculated after correction
for loading differences with GAPDH. These values are shown below each blot.

Nuclear SREBPs Activate Unsaturated Fatty Acid Biosynthesis

35303

TABLE II
Stearoyl-CoA desaturase activities in livers of wild-type andtransgenic mice
Each value represents the mean 6 S.E. Liver microsomes were prepared from the indicated mice described in Experiments A and B of
Table I. Microsomal protein (100 mg) was incubated with a reaction
mixture containing [1-14C]stearoyl-CoA for 5 min. The products were
saponified, acidified, and the fatty acids were extracted and separated
by TLC as described under “Experimental Procedures.”[1-14C]Stearoyl-CoA 3 Oleate

Genotype of mice

nmol z min21 z mg protein21

FIG. 3. Linearity of signals for SCD1 mRNA in wild-type andtransgenic mouse livers using the RNase protection assay. 32PLabeled cRNA probe for mouse SCD1 was hybridized to the indicatedamount of total RNA from wild-type or transgenic mouse livers for 10
min at 68 °C. Protected fragments were separated by gel electrophoresis and exposed to film for 30 min at 280 °C with an intensifying screen(top). The gel was also quantified as described in the legend to Fig. 1,
and the results are plotted in the lower panel. Levels of SCD1 mRNA
are expressed in relation to the signal generated by 2.5 mg of RNA in
wild-type liver, which is arbitrarily set at 1.

Experiment A
Wild-typeTgSREBP-1aExperiment B
Wild-typeTgSREBP-1c
TgSREBP-2
a

0.9 6 0.1
5.0 6 0.3a
0.6 6 0.1
1.5 6 0.1a
1.5 6 0.1a

p , 0.001 between transgenic and wild-type mice (Student’s t test).

TABLE III
Fatty acid compositions of livers in wild-type and transgenic mice
Liver samples from each mouse were extracted, and the major classesof lipids were separated on a silica column. The lipid fractions were
methyl esterified and quantified by GLC as described under “Experimental Procedures.” Each value represents the mean from 6 mice inExperiment A and 5 mice in Experiment B. Standard errors for all
values were less than 20% of the mean and are omitted from the tablefor clarity. Bold values denote a level of statistical significance of p ,0.01 between wild-type and transgenic mice (Student’s t test).
Fatty acid composition (% of total)
Genotype of mice

FIG. 4. Amounts of mRNA encoding SCD1 and SCD2 in livers
from wild-type and transgenic mice as measured by the RNase
protection assay. Total RNA was isolated from wild-type epididymal
fat and 5 mg was hybridized with a 32P-labeled cRNA probe for SCD1
(lane 1) or SCD2 (lane 2) as described under “Experimental Procedures.” White fat contains mRNA transcripts for both SCD1 and SCD2,
and therefore, was used as a positive control. Total RNA from livers ofthe mice described in Table I was isolated and equal aliquots (5 mg)
were hybridized with a 32P-labeled cRNA probe for SCD1 (lanes 3–7) orSCD2 (lanes 8 –12). The protected fragments were separated by gelelectrophoresis and exposed to film for 30 min at 280 °C with anintensifying screen for SCD1 in liver and for 12 h at room temperature
for white fat and SCD2 in liver.

we measured SCD activity in liver microsomes from wild-type
and transgenic mice. Table II shows the mean SCD activities asestimated 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 inlivers from TgSREBP-1c and TgSREBP-2 mice were both approximately 2.5-fold higher than wild-type control levels.
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

14:0

16:0

16:1

18:0

18:1

18:2

20:4

22:6

Total fatty acids
Wild-typeTgSREBP-1a

0.5
0.4

22
13

2.4
7.1

12
3.0

18
65

17
3.0

16
2.8

5.5
0.7

Wild-typeTgSREBP-1c
TgSREBP-2

0.2
0.5
0.5

21
23
16

1.4
4.3
4.3

14
6.8
7.3

15
32
33

16
13
17

18
9.8
10

5.8
3.4
2.9

Cholesteryl esters
Wild-typeTgSREBP-1a

0.7
0.4

53
13

3.9
7.6

5.9
1.1

15
72

6.6
2.7

2.3
0.3

0
0

Wild-typeTgSREBP-1c
TgSREBP-2

0.4
0.7
0.7

46
27
17

2.3
5.2
5.9

6.2
1.9
1.8

19
46
50

9.0
11
15

3.0
0.9
1.7

0
0
0

Triglycerides
Wild-typeTgSREBP-1a

1.0
0.4

22
11

4.9
9.8

2.9
0.8

37
69

21
4.2

2.9
0.5

0
0

Wild-typeTgSREBP-1c
TgSREBP-2

0.5
0.7
0.8

20
22
14

2.8
6.7
6.2

3.3
1.2
1.1

35
44
41

23
17
22

3.6
1.5
2.9

0
0
0

Phospholipids
Wild-typeTgSREBP-1a

0
0

21
13

1.5
2.0

13
19

12
24

15
4.3

20
23

6.6
5.3

Wild-typeTgSREBP-1c
TgSREBP-2

0
0
0

23
22
17

0.9
1.6
1.5

18
15
16

11
14
17

14
11
13

18
20
19

5.1
6.5
5.4

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.
The hepatic cholesteryl ester fractions from all three lines oftransgenic mice contained a 2-fold increase in the relative
amount of palmitoleic acid (16:1), while the relative amount ofoleic acid (18:1) was 4.8-fold higher in TgSREBP-1a mice and35304

Nuclear SREBPs Activate Unsaturated Fatty Acid Biosynthesis
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 increasedby 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 thatmeasured in the cholesteryl ester fractions.
DISCUSSION

FIG. 5. The ratio of palmitoleic (16:1) to palmitic (16:0) andoleic (18:1) to stearic (18:0) acids in various lipid fractions extracted from livers of wild-type and transgenic mice. Total lipids
were extracted from livers of wild-type and transgenic mice described inTable I. Lipid fractions were separated on a silica column, methyl
esterified, and quantified by GLC as described under “Experimental
Procedures.” The values used for calculating the ratio of monounsaturated to saturated fatty acids for wild-type mice are the means of thewild-type mice in Experiments A and B presented in Table III. The
ratios for the transgenic mice are derived directly from the means
presented in Table III. The ratios of palmitoleic (16:1) to palmitic (16:0)
acids in total lipid extracts (panel A), cholesteryl esters (panel B), andtriglycerides (panel C) from livers of wild-type and TgSREBP-1a, -1c,and -2 mice are shown. The ratio of oleic (18:1) to stearic (18:0) acid foreach lipid fraction is presented in panels D-F.

2.5-fold higher in TgSREBP-1c and -2 mice (Table III). Thechanges measured in the relative amounts of monounsaturated
and saturated fatty acids in the triglyceride fractions fromlivers of the TgSREBP-1a, -1c, and -2 mice are similar to thechanges measured in the cholesteryl esters. Inasmuch as thecholesteryl ester and triglyceride content in livers of TgSREBP-1a and TgSREBP-2 mice are significantly elevated (seeTable I), the absolute monounsaturated fatty acid content ineach fraction is dramatically increased. Fatty acid compositional changes in the phospholipid fractions were much lessstriking, 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

The purpose of the current study was to gain insight into theeffects 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 theincorporation 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 notbeen 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 thesemice.
Most of the carbon source for fatty acid synthesis comes frompyruvate, 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 forlipid 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, andeach is estimated to contribute approximately 50% in rat liver(41). The first reaction is the oxidative decarboxylation ofmalate to form pyruvate and CO2. This reaction is catalyzed bymalic enzyme, which simultaneously generates NADPH from
NADP2. 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 elevatedmRNAs 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 activatesthe genes responsible for the production of the acetyl-CoA andNADPH required for fatty acid synthesis. Since lipogenesis isglobally stimulated in TgSREBP-1a livers, it is possible thattranscription of the genes encoding the enzymes responsible forthe production of citrate are also increased (i.e. glucokinase,phosphofructokinase I, and pyruvate kinase), although thesemRNAs were not studied in the current experiments.
To date, SREBP-1a has been shown to directly bind to thepromoters and activate the transcription of ACC, FAS, andGPAT (9 –11). To our knowledge, the promoters for ATP citratelyase, malic enzyme, Glu-6-PD, and PGDH have not been studied. Therefore, we cannot conclude SREBP-1a activates thetranscription of these genes directly. However, whether theeffect 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.

Nuclear SREBPs Activate Unsaturated Fatty Acid Biosynthesis
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 miceexpressing SREBP-1a and SREBP-2, indicating that the blockin 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 anincrease in the measured SCD activity in liver microsomes(Table II). This is of possible metabolic importance because thepredominant 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 andincreases 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 acidprofile was measured in the triglyceride fractions. Approximately 80% of the fatty acids present in these fractions weremonounsaturated with the vast majority being oleic acid. Thechanges in liver cholesteryl ester and triglyceride fatty acidcomposition 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 iscurrently under investigation.
The changes measured in the mRNAs for fatty acid synthetic
enzymes in livers of TgSREBP-1c and TgSREBP-2 mice were ina 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 theirability to activate the lipogenic genes GPAT and S14. NeithermRNA was changed in TgSREBP-2 mouse livers, but both weresignificantly increased in TgSREBP-1c and -1a livers. In thecase of GPAT, this finding is consistent with GPAT promoterreporter 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 theidea that SREBP-1 is relatively selective as compared with
SREBP-2 in activating lipogenic genes (18). The molecularbasis 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 acidin 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

2
J. D. Horton, H. Shimano, J. L. Goldstein, and M. S. Brown, unpublished observations.

35305

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 potentialof high-level expression of nuclear SREBPs to activate theprograms 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 arefound to block the activities of nuclear SREBPs in liver or tomodify the target genes so they lose their ability to respond to
SREBPs.
Acknowledgments—We thank Drs. Michael S. Brown and Joseph L.
Goldstein for their continued encouragement and critical reading of themanuscript. 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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