Activation of Cholesterol Synthesis in Preference to Fatty Acid Synthesis in
Liver and Adipose Tissue of Transgenic Mice Overproducing Sterol Regulatory
Element-binding Protein-2
Jay D. Horton,* Iichiro Shimomura,* Michael S. Brown,* Robert E. Hammer,‡§ Joseph L. Goldstein,* and Hitoshi Shimano**Department of Molecular Genetics, ‡Department of Biochemistry, and §Howard Hughes Medical Institute, University of Texas
Southwestern Medical Center, Dallas, Texas 75235

Abstract
We produced transgenic mice that express a dominant-positive truncated form of sterol regulatory element-binding
protein-2 (SREBP-2) in liver and adipose tissue. The encoded protein lacks the membrane-binding and COOH-terminal regulatory domains, and it is therefore not susceptible
to negative regulation by cholesterol. Livers from the transgenic mice showed increases in mRNAs encoding multiple enzymes of cholesterol biosynthesis, the LDL receptor, and fatty acid biosynthesis. The elevations in mRNAfor 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA)synthase and HMG CoA reductase were especially marked(13-fold and 75-fold, respectively). As a result, the transgenic livers showed a 28-fold increase in the rate of cholesterol synthesis and a lesser fourfold increase in fatty acidsynthesis, as measured by intraperitoneal injection of[3H]water. These results contrast with previously reported
effects of dominant-positive SREBP-1a, which activatedfatty acid synthesis more than cholesterol synthesis. In adipose tissue of the SREBP-2 transgenics, the mRNAs forcholesterol biosynthetic enzymes were elevated, but themRNAs for fatty acid biosynthetic enzymes were not. We
conclude that SREBP-2 is a relatively selective activator ofcholesterol synthesis, as opposed to fatty acid synthesis, inliver and adipose tissue of mice. (J. Clin. Invest. 1998. 101:
2331–2339.) Key words: cholesterol • low density lipoprotein •sterol regulatory element binding proteins • fatty acids •transgenic mice

Introduction
Cholesterol and fatty acids, the primary lipids synthesized inliver, are subject to different patterns of regulation (reviewed
in references 1 and 2). Cholesterol synthesis is high when animals are fed a cholesterol-free diet, and it declines dramatically when cholesterol is fed. Cholesterol synthesis is also enhanced when the demand for cholesterol is increased, as when

Address correspondence to Joseph L. Goldstein, Department of Molecular Genetics, University of Texas Southwestern Medical Center,5323 Harry Hines Blvd., Room L5-238, Dallas, TX 75235. Phone:214-648-2141; FAX: 214-648-8804.
Received for publication 2 February 1998 and accepted in revised
form 17 March 1998.
J. Clin. Invest.
© The American Society for Clinical Investigation, Inc.
0021-9738/98/06/2331/09 $2.00
Volume 101, Number 11, June 1998, 2331–2339
http://www.jci.org

animals consume bile acid–binding resins. In contrast, fatty
acid synthesis is driven primarily by the availability of carbohydrate and the stimulating action of insulin. Despite these different patterns of regulation, recent evidence suggests thatboth biosynthetic pathways can be regulated by a common
family of transcription factors designated sterol regulatory element binding proteins (SREBPs)1 (reviewed in reference 3).
SREBPs are members of a broad class of transcription activators that use basic-helix-loop-helix-leucine zipper (bHLH-Zip)
motifs to dimerize and to bind DNA. SREBPs are unique inthat they are synthesized as membrane-bound precursors thatmust be liberated by proteolysis in order to enter the nucleus(3). They are also unique in having a tyrosine in place of anotherwise conserved arginine, which allows them to bind tononpalindromic sterol regulatory elements in preference toclassic palindromic E boxes (4).
The membrane-bound precursor forms of SREBPs containz 1,150 amino acids. The NH2-terminal segment of z 480
amino acids includes the bHLH-Zip domain and an acidictranscription activating domain at the extreme NH2 terminus.
The bHLH-Zip domain is followed by a membrane attachment domain of z 80 amino acids that consists of two membrane-spanning segments separated by a short hydrophilic
loop. This is followed by a COOH-terminal regulatory domainof z 590 amino acids. The SREBPs are oriented so that theirNH2-terminal and COOH-terminal domains project into thecytoplasm, and the 31-amino acid loop projects into the lumenof the endoplasmic reticulum (3).
The mechanism for regulation of SREBP activity was elucidated through studies of cultured fibroblasts. When such cells
are deprived of sterols, a two-step proteolytic process releases
the NH2-terminal domains of the SREBPs so that they can enter the nucleus (3). There they bind to sterol regulatory elements in the promoter regions of genes encoding enzymes ofcholesterol biosynthesis (HMG CoA reductase, HMG CoA
synthase, farnesyl diphosphate synthase, squalene synthase,and others) (5–8). The SREBPs also bind to regulatory sequences in the promoters of at least two genes involved in fatty
acid synthesis, acetyl CoA carboxylase and fatty acid synthase(4, 9, 10), and at least one gene involved in triglyceride synthesis, glycerol-3-phosphate acyltransferase (11). Binding to allthese promoters leads to transcriptional activation, which results in increased synthesis of cholesterol and fatty acids.
When sterols overaccumulate in cells, the proteolytic release

1. Abbreviations used in this paper: bHLH-Zip, basic-helix-loophelix-leucine zipper; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HMG CoA, 3-hydroxy-3-methylglutaryl coenzyme A; PEPCK,
phosphoenolpyruvate carboxykinase; PPAR-g, peroxisomal proliferator-activated receptor; SREBP, sterol regulatory element-binding
protein.
Transgenic Mice Expressing Truncated SREBP-2

2331

of SREBPs is blocked, the NH2-terminal fragments remainbound to membranes, and transcription of all target genes declines (3).
Three SREBPs are currently recognized (3). Two of these,
designated SREBP-1a and SREBP-1c, are produced from asingle gene through the use of alternate promoters (3, 12).
SREBP-1c was cloned independently as a protein that binds toE-boxes and was named ADD-1 (13). SREBP-1a and -1c differ in the length of the first exon, which encodes a portion of
the acidic transcription activating domain. This domain ismuch longer in SREBP-1a than it is in SREBP-1c (12). The
other member of the family, designated SREBP-2, has a longtranscription activation domain resembling that of SREBP-1a(6). Livers of mice, humans, and hamsters produce relatively
large amounts of SREBP-1c and smaller amounts of SREBP-2
and -1a (12, 14).
The reason for the three isoforms of SREBP is not yet
known. The proteins appear to act primarily as homodimers,
and heterodimerization is not required for their activity (reference 6 and Pai, J.-T., M.S. Brown, and J.L. Goldstein, unpublished observations). All three SREBPs are capable of activating all of the known target genes, but they do so with different
efficiencies. In general, SREBP-1a and SREBP-2 are much
more active than SREBP-1c (reference 15 and Pai, J.-T., M.S.
Brown, and J.L. Goldstein, unpublished observations).
In an attempt to decipher the roles of individual SREBPs
in liver, we have produced transgenic mice that overexpress
truncated dominant-positive forms of SREBP-1a (16) or
SREBP-1c (15). The encoded proteins terminate at a point between the bHLH-Zip domain and the membrane-attachment
domain. Such proteins are never membrane bound, and they
enter the nucleus directly without a requirement for proteolysis. As a result, these proteins are not down-regulated whencholesterol accumulates. Transgene expression is driven by thepromoter for phosphoenolpyruvate carboxykinase (PEPCK),
which is expressed in liver, adipose tissue, and kidney. Expression is induced to high levels in the liver when the mice consume a low carbohydrate/high protein diet (15, 16).
Transgenic animals expressing dominant-positive SREBP-1a
exhibited a striking phenotype. Their livers were enlarged up
to fourfold, owing to the accumulation of massive amounts oftriglyceride and cholesteryl ester (16). Despite the buildup ofthese end-products, the mRNAs encoding multiple enzymes in
the cholesterol and fatty acid biosynthetic pathways weremarkedly elevated. These include the mRNAs for HMG CoA
reductase, HMG CoA synthase, farnesyl diphosphate synthase, squalene synthase, acetyl CoA carboxylase, fatty acidsynthase, and stearoyl CoA desaturase. The mRNA encoding
the LDL receptor was also increased. As a result, the livers ofthese animals massively overproduced cholesterol and fatty acids as measured by the rate of incorporation of intraperitoneally injected [3H]water.
The white adipose tissue of the SREBP-1a transgenic mice
was normal in young animals, but it involuted as the animals
became older (16). By the age of 4 mo, the amount of white
adipose tissue had declined by . 90%, as reflected by theamount of epididymal fat.
In contrast to the striking phenotype of the SREBP-1a
mice, we observed a much milder phenotype in animals expressing equivalent amounts of the truncated form of SREBP-1c(15). In these animals the liver was not significantly enlarged.
The organ showed a modest accumulation of triglyceride and a
2332

Horton et al.

modest increase in the mRNAs encoding the fatty acid biosynthetic enzymes. The mRNAs encoding the cholesterol biosynthetic enzymes were not elevated.
In the current study we have extended these observations
to SREBP-2. We have produced transgenic mice that overexpress a truncated dominant-positive form of SREBP-2 undercontrol of the PEPCK promoter. The pathways of cholesterol
biosynthesis and fatty acid biosynthesis are both activated in
the SREBP-2 transgenic animals, but the ratio of cholesterol
biosynthesis relative to fatty acid biosynthesis is much higher
in these animals than in the SREBP-1a transgenics. This is attributable to a marked increase in the mRNAs for HMG CoA
synthase and HMG CoA reductase. These data indicate thatthe major function of SREBP-2 is to activate cholesterol biosynthesis in preference to fatty acid synthesis in liver and adipose tissue.

Methods
Materials and general methods. Sequencing reactions were performedon an Applied Biosystems 373A DNA Sequencer (Foster City, CA)
by the dideoxy chain termination method. The content of cholesterol
and triglyceride in plasma and liver was measured as described previously (16). Transgenic mice expressing SREBP-1c436 (encoding
amino acids 1–436 of human SREBP-1c) (15) and SREBP-1a460, line
B (encoding amino acids 1–460 of human SREBP-1a) (16) were described in the indicated reference.
Measurement of hepatic unesterified and esterified cholesterol.
Total lipids were extracted from 200–250-mg aliquots of liver. Unesterified and esterified cholesterol were separated on 500-mg silica
columns (Cat no. 1211-3036; Varian, Harbor City, CA) as described(17) and quantified by gas liquid chromatography (18).
Plasmid construction. The expression plasmid pPEPCK-SREBP2(1–468) contained the rat PEPCK promoter (2.4 kb) fused to a
cDNA encoding amino acids 1–468 of human SREBP-2 (6) followedby two consecutive stop codons. It was constructed as previously described for pPEPCK-SREBP-1a460 except that the cDNA fragment
was produced by PCR and ligated into the SmaI site of the pCMVPEPCK promoter (16). The PCR reaction was performed using
pSREBP-2 (6) as a template, Pfu polymerase, a 59 primer corresponding to bp 2 74 to 2 45 in the 5 9 untranslated region ofhuman SREBP-2 (59-GGGCGGTGGCGACGGCACCGCCCCCGCGTCT-39), and a 39 primer corresponding to amino acids 459–468
followed by two consecutive stop codons (59-GGGTCATCAGTCTGGCTCATCTTTGACCTTTGCATCATC-39). The integrity of
the construct was confirmed by DNA sequencing.
Production and maintenance of transgenic mice. Techniques used
for generating transgenic mice were previously described (16). A total of 1,502 fertilized eggs that were microinjected with the SalI-SphI
fragment of pPEPCK-SREBP-2(1–468) survived to the two-cellstage. Among the 184 offspring, 23% had integrated the transgene asdetermined by dot blot hybridization of DNA from tail homogenates.
Of 42 DNA-positive mice subjected to partial hepatectomy, 30% produced truncated SREBP-2 as determined by immunoblot analysis.
Mice with high levels of transgene expression in liver were bred to
C57BL/6J 3 SJL F1 mice, and two independent lines of SREBP2(1–468) mice were established, designated lines A and B. To obtainhigher expression, hemizygotes of line B were mated to produce homozygotes for the transgene. Hemizygous mice from line A and homozygous mice from line B were used for all studies. Mice werehoused in colony cages and maintained on a 14-h light/10-h dark cycle. Transgenic mice and nontransgenic littermates were fed a low
carbohydrate/high protein diet (No. 5789C-3) from Purina Mills Inc.
(St. Louis, MO) containing 71% (wt/wt) casein and 4.25% sucrose for
2 wk to induce maximal expression of the transgene (19). All mice
were fasted 4 h before death during the early phase of the light cycle.

Immunoblotting. Nuclear extracts were prepared from pooled
liver samples as previously described (16). Each sample (30 mg protein) was subjected to 8% SDS-PAGE, electrophoretically transferred to Hybond C extra membranes (Amersham Corp., Arlington
Heights, IL), and incubated with 5 mg/ml of rabbit anti–human
SREBP-2 IgG, which was raised against amino acids 48–403 of human SREBP-2 (6). Immunoblot analysis was carried out with the Enhanced Chemiluminescence (ECL) Western Blotting Detection System Kit (Amersham Corp.) with a horseradish peroxidase-labeled
donkey anti–rabbit IgG antibody (Amersham Corp.) as previously
described (16).
Blot hybridization of RNA. Total RNA was prepared frommouse liver and epididymal fat pad using the RNeasy™ Total RNA
kit (Qiagen Inc., Chatsworth, CA) and the acid guanidinium thiocyanate-phenol-chloroform method (20), respectively. Equal aliquotsof total RNA from each group of mice were pooled (total, 15 mg) andsubjected to Northern blot analysis with the indicated cDNA probes
prepared as previously described (15, 16, 21). A HindIII fragment(1.3 kb) of the human SREBP-2 cDNA was used as a cDNA probe
for the transgene. A probe for mouse PPAR-g (22) was prepared byreverse transcriptase-PCR using mouse liver poly(A)1 RNA as
template as previously described for other probes (16). The 59 and39 primers were 59-ACAAGACTACCCTTTACTGAAATTACCATG-39 and 59-GGCACTTCTGAAACCGACAGTACTGACATTTATTT-39, respectively. The bands on the Northern blots were
quantified by exposure of the filter to a BAS1000 Fuji PhosphorImager (Tokyo, Japan), and the results were normalized to the signal
generated from GAPDH mRNA.
RNase protection assay. cRNA probes were prepared from
cDNA fragments for human SREBP-1 and SREBP-2 that were produced by PCR amplification using the plasmids pPEPCK-SREBP1a460 (16) and pPEPCK-SREBP-2(1–468) (this paper) as templates,

respectively. The following primers were used. Human SREBP-1:
59 primer, 59-GGCACAGACCCTGCCAGCC-39; and 39 primer,
59-CTGGGGTAGCCTAACACAGG-39 (5). Human SREBP-2: 59
primer, 59-GTAGCAGCAGCGGCAGCAGTGG-39; and 39 primer,
59-AAAGGCTGCTGGATGATCCTCG-39 (6). The cDNA fragment for human SREBP-1 is identical in DNA sequence for isoforms
SREBP-1a and -1c. HindIII and EcoRI sites were added to both 59
and 39 primers, respectively. The RNase protection assay was done aspreviously described (12, 14). For comparison of transgene expression levels in livers of SREBP-1a, -1c, and -2 transgenic mice, eachprobe was designed so that each protected fragment would containthe same number of 32P atoms (i.e., 69).
Cholesterol and fatty acid synthesis in vivo. Five 12-wk-old micehomozygous for the human SREBP-2 transgene (line B) and wildtype mice from the same litters (three females and one male for eachgroup) were fasted for 4 h and killed during the early light cycle. The
rates of incorporation of intraperitoneally injected [3H]water intocholesterol and fatty acid were measured in liver as previously described (16).

Results
To produce transgenic mice expressing a dominant-positive
form of SREBP-2, we constructed a plasmid encoding aminoacids 1–468 of human SREBP-2 under control of the PEPCK
promoter. The encoded protein includes the bHLH-Zip DNA
binding domain and the NH2-terminal acidic transactivation
domain, but it lacks the membrane-attachment domain and
the COOH-terminal regulatory domain. Previous studies intissue culture demonstrated that such truncated SREBPs enter

Figure 1. Photograph oflivers from 12-wk-old
wild-type mice and transgenic mice expressing
truncated dominant-positive SREBP-1a, -1c, and-2. The SREBP-1a (lineB) and SREBP-1c mice
were described in references 15 and 16. The
SREBP-2 mouse wasfrom line B. All animals
were fed the low carbohydrate/high protein
diet for 2 wk before killing. (Lower panel) schematic diagram of thedomain structure of
SREBP-1a, -1c, and -2,
illustrating the site oftruncation used for construction of the transgenes. TM, transmembrane segments.
Transgenic Mice Expressing Truncated SREBP-2

2333

Table I. Phenotypic Comparison of Wild-type and TgSREBP-2 Mice
TgSREBP-2 miceParameterWild-type mice

Number and sex
Body weight (grams)
Liver weight (grams)
Liver weight/body weight
Epididymal fat pad weight (grams)
Epididymal fat weight/body weight
Liver cholesterol content (mg/gram)
Liver triglyceride content (mg/gram)Total plasma cholesterol (mg/dl)
Total plasma triglycerides (mg/dl)

Five males
27.360.5
1.560.11
0.05360.003
0.2460.04
0.00960.001
2.260.09
7.261.4
8766.4119617

Line A

Five males
27.161.0
1.560.03
0.05660.002
0.4060.06
0.01560.002
3.660.17 (P , 0.001)*
10.762.711164.0 (P , 0.05)*
101612

Line B

Five males
24.961.1
1.560.09
0.05860.002
0.2760.02
0.01160.001
5.360.57 (P , 0.01)*
28.366.9 (P , 0.05)*
7068.5 (P , 0.05)*
6365.5

Each value represents the mean6SEM of five 16-wk-old male mice. Wild-type and line A hemizygous transgenic mice were obtained from the sametwo litters. Line B transgenic mice were homozygous for the transgene and obtained from four littermates from hemizygote matings. All mice were
fed the low carbohydrate/high protein diet for 2 wk and fasted 4 h before death. *Values in parentheses denote the level of statistical significance(Student’s t test) between wild-type and the indicated line of transgenic mice. For parameters in which the level of significance is P . 0.05, no values
are shown.

the nucleus directly without a requirement for proteolysis (23).
The site of truncation in SREBP-2 is equivalent to the sites oftruncation in the SREBP-1a and -1c proteins that have beenstudied previously in transgenic animals (Fig. 1, lower panel).
The SREBP-2 transgene construct was injected into mouseeggs, and we derived two independent lines of transgenic mice,designated A and B. To increase expression, mice from line B
were bred to homozygosity for the transgene. The line A mice
were maintained in the hemizygous state. Fig. 1 (upper panel)compares the appearance of the liver of one representative
mouse from SREBP-2, line B and livers from previously described transgenic mice expressing dominant-positive forms of
SREBP-1a and -1c (15, 16). As reported previously, the livers
from the SREBP-1a animals were grossly enlarged and pale,owing to massive accumulation of cholesteryl esters and triglycerides (16), whereas the SREBP-1c livers were generally indistinguishable from wild-type (15). Livers from the SREBP-2
animals were not visibly enlarged, but they exhibited a distinct
pale color that was consistently different from that of wild-typemice (Fig. 1, upper panel).
Table I compares various phenotypic parameters in wildtype mice and SREBP-2 transgenic mice of lines A and B. Statistically significant changes were confined to the liver andplasma. Livers from the SREBP-2 transgenic animals werenormal in weight, but they contained increased amounts ofcholesterol and triglyceride. The levels of both lipids werehigher in line B than in line A. The plasma cholesterol level
was increased slightly in the line A animals and decreasedslightly in the line B animals, a finding of questionable biologic
significance. Plasma triglyceride levels were reduced in thetransgenic mice, but this was not statistically significant, owing
to the large variation in triglyceride levels in the wild-type
mice. Importantly, the weight of adipose tissue, as reflected in
the epididymal fat pad, was normal in the SREBP-2 transgenic
mice, a finding that contrasts with the marked decline observed in the SREBP-1a transgenics (16).
Livers from line B mice expressed a somewhat higher levelof truncated SREBP-2 protein as compared with livers from
2334

Horton et al.

the line A animals (1.3-fold) as determined by immunoblotting(Fig. 2). Similar results were seen in one other experiment inwhich these levels were compared.
In the Northern blotting experiment of Fig. 3, the livers of
the line B mice contained about threefold more transgene
SREBP-2 mRNA than the livers of the line A mice (Fig. 3, upper left panel). A similar trend was seen when we examined
levels of mRNA for putative SREBP target genes. Elevations
were observed in the levels of mRNA for endogenous SREBP-1
and -2, the LDL receptor, and multiple genes of cholesterol
biosynthesis, including HMG CoA synthase, HMG CoA reductase, farnesyl diphosphate (FPP) synthase, and squalene
synthase. The magnitude of the increase in HMG CoA reductase mRNA is particularly noteworthy (16-fold in line A and
75-fold in line B). Elevated levels of mRNAs for fatty acid biosynthetic enzymes were also observed, including acetyl CoA
carboxylase, fatty acid synthase, and stearoyl CoA desaturase.
In each case the elevation was greater for the line B animals ascompared with the line A animals. We also observed an elevation in the mRNA encoding the microsomal triglyceride transfer protein (MTP), which participates in the synthesis of tri-

Figure 2. Immunoblot analysis of
truncated human SREBP-2 in nuclear
extracts from livers of two lines oftransgenic SREBP-2 mice (A and B).
Livers from the mice described in Table I were pooled, and aliquots of thepooled nuclear extracts (30 mg protein) were subjected to SDS-PAGE
and transferred electrophoretically toa nitrocellulose filter. Immunoblot
analysis was carried out with 5 mg/ml
rabbit anti–human SREBP-2 IgG as the primary antibody, which
does not recognize endogenous mouse SREBP-2. Bound antibodies
were visualized as described in Methods. Filters were exposed to film
for 15 s.

Figure 3. Amounts of various mRNAs in livers of wild-type (WT) mice and two lines of transgenic SREBP-2 mice (A and B) as measured byblot hybridization. Total liver RNA isolated from mice described in Table I was pooled (five mice per group), and 15-mg aliquots were subjected
to electrophoresis and blot hybridization with the indicated 32P-labeled probe and with a control 32P-labeled probe directed against GAPDH.
The amount of radioactivity in each band was quantified as described in Methods. The fold increase in each mRNA of transgenic mice, relative
to that of wild-type control mice, was calculated after correction for loading differences with GAPDH. These values are shown below each blot.
The probe for stearoyl CoA desaturase (SCD) was the mouse SCD-1 cDNA fragment that can detect both SCD-1 and SCD-2 mRNAs (16).
FPP, farnesyl diphosphate; MTP, microsomal triglyceride transfer protein.

Figure 4. Amount of various mRNAs in white adipose tissues of wild-type (WT) and two lines of transgenic SREBP-2 mice (A and B) as measured by blot hybridization. Total RNA isolated from epididymal fat pads of mice described in Table I was pooled, and 15-mg aliquots were subjected to electrophoresis and blot hybridization with the indicated 32P-labeled probe and with a control 32P-labeled probe directed against
GAPDH. The amount of radioactivity in each band was quantified as described in Methods. The fold increase in each mRNA of transgenic mice,relative to that of wild-type control mice, was calculated after correction for loading differences with GAPDH. These values are shown below
each blot. The probe for stearoyl CoA desaturase (SCD) was the mouse SCD-1 cDNA fragment that can detect both SCD-1 and SCD-2 mRNAs(16). FPP, farnesyl diphosphate.
Transgenic Mice Expressing Truncated SREBP-2

2335

Table II. In Vivo Synthesis of Cholesterol and Fatty Acids in Livers from Wild-type and TgSREBP-2 Mice
Incorporation of [3H]water into
Genotype of mice

Wild-type (n 5 5)
TgSREBP-2 (n 5 5)

Liver weight

Digitonin-precipitable sterols

Fatty acids

grams

mmol/h per gram

mmol/h per organ

mmol/h per gram

mmol/h per organ

1.0460.04
1.3760.07

0.3560.1
9.961.2

0.3660.1
13.461.2

4.060.4
15.362.9

4.160.5
21.765.1

Each value represents the mean6SEM of four female and one male 12-wk-old littermate mice of the indicated genotype. The transgenic SREBP-2
mice were from line B. All mice were fed the low carbohydrate/high protein diet for 2 wk and were fasted 4 h before the experiment. [3H]water was
injected intraperitoneally, and 1 h later the liver was removed for measurement of its content of 3H-labeled digitonin-precipitable sterols and fatty
acids as described in Methods. All differences between wild-type and transgenic SREBP-2 mice were statistically significant by the Student’s t test
(P , 0.02).

glyceride-rich VLDL (24). Inasmuch as this gene has not beenstudied previously as a target for SREBPs, we do not knowwhether the elevation is a direct effect of SREBP-2 action orwhether it is secondary to the increased lipid content of theliver. The mRNA for cholesterol 7-a hydroxylase was elevatedslightly in the line B animals. In agreement with previous observations with the other transgenic SREBP mice (15, 16), we
found no elevation in the levels of mRNA for apoproteins of
the plasma lipoprotein transport system (namely, apo AI, apo
B, and apo E) in livers of the mice expressing SREBP-2.
The PEPCK promoter that drives expression of the
SREBP-2 transgene is active in adipose tissue as well as inliver. In the experiment of Fig. 4, we compared the levels of
various mRNAs in epididymal fat pads of wild-type mice andtransgenic lines A and B. The amount of transgene expression
was higher in the line B animals than in the line A animals, andthis was reflected in elevated levels of mRNA for the LDL receptor and the cholesterol biosynthetic enzymes. Surprisingly,
we did not observe consistent elevations in mRNAs for acetyl
CoA carboxylase, fatty acid synthase, or stearoyl CoA desaturase in adipose tissue. We also did not observe significant effects on mRNAs for relatively fat-specific genes such as lipoprotein lipase, leptin, or PPAR-g. Thus, in adipose tissue thetruncated SREBP-2 has a relatively specific stimulatory effecton transcription of genes involved in cholesterol supply as opposed to fatty acid input.
In livers of the SREBP-2 transgenic mice, the increases in
mRNAs encoding lipogenic enzymes were reflected in elevated rates of lipid synthesis (Table II). To make these measurements, wild-type and SREBP-2 transgenic mice (line B)
were injected intraperitoneally with [3H]water, and the incorporation into digitonin-precipitable sterols and fatty acids wasmeasured in liver extracts 1 h later. These studies revealed a
massive increase in sterol synthesis (28-fold) and a lesser increase in fatty acid synthesis (fourfold) in the SREBP-2 transgenics when expressed on the basis of liver weight. The increase in cholesterol synthesis was associated with an 11-foldincrease in cholesteryl ester content in the SREBP-2 transgenic livers (Fig. 5).

Discussion
The current experiments complete a series in which dominantpositive truncated versions of all three SREBPs were expressed in liver and adipose tissue of transgenic mice under
2336

Horton et al.

control of the PEPCK promoter. Figs. 6–9 compare the resultswith respect to the liver. The experiments in each strain were
performed at different times, but the animals were all approximately the same age, and they were all fed the low carbohydrate/high protein diet for 2 wk to induce transgene expression
before killing. Fig. 6 compares the levels of transgene expression as determined by RNase protection assays using probesthat were adjusted to contain the same number of 32P atoms ineach protected fragment. The level of transgene-encoded
mRNA was higher for SREBP-1a than for SREBP-1c or
SREBP-2. Although these differences preclude a direct comparison of each isoform’s absolute effect on the absolute rateof lipid synthesis, they do allow a valid comparison of the relative effect of each isoform on cholesterol synthesis versus triglyceride synthesis.
Although all three SREBP isoforms tended to stimulate
expression of multiple genes involved in lipid synthesis, therelative degree of stimulation varied in ways that indicated differential effects on the pathways of cholesterol as opposed tofatty acid synthesis. As shown in Fig. 7, SREBP-2 was more effective than SREBP-1a in increasing the amounts of the three

Figure 5. Content of unesterified (A) and esterified (B) cholesterol inlivers of wild-type and transgenic SREBP-2, line B mice. Four 16-wkold wild-type female mice and five transgenic SREBP-2, line B littermate female mice were fed the low carbohydrate/high protein diet for
2 wk and fasted 4 h before study. Aliquots of liver (200–250 mg) weretaken for measurement of unesterified and esterified cholesterol as
described in Methods. Each circle denotes the value from an individual animal, and the bar represents the mean.

Figure 6. Relative amounts
of mRNA encoding truncated SREBP-1c (lane A),
SREBP-1a (lane B), and
SREBP-2 (lane C) in livers
from transgenic mice asmeasured by RNase protection. Total RNA fromthe livers of mice expressing the truncated transgenes encoding SREBP-1c(15), SREBP-1a (16), and
SREBP-2, line B (this paper) was isolated, and equal
aliquots (10 mg) were hybridized at 688C to the appropriate 32P-labeledcRNA probe as describedin Methods. Protected fragments were separated bygel electrophoresis and exposed to film for 8 h at
2808C with an intensifying
screen. The radioactivity inthe gels was quantified as
described in Methods, normalized to the b-actin signal, and expressed as thefold change relative to thevalue in SREBP-1c mice,
which was arbitrarily set
at 1.0.

mRNAs of the cholesterol biosynthetic pathway (HMG CoA
synthase, HMG CoA reductase, and squalene synthase),
whereas SREBP-1a was more effective than SREBP-2 with respect to the two mRNAs of fatty acid biosynthesis (acetyl CoA
carboxylase and fatty acid synthase). Both SREBPs were approximately equipotent with respect to the LDL receptor.
SREBP-1c was much less effective than either SREBP-1a or
SREBP-2 in stimulating transcription of all of the genes.
The differential effects of the three SREBPs on mRNAtranscription were reflected in differential rates of lipid synthe-

sis from [3H]water (Fig. 8). SREBP-2 stimulated sterol synthesis more profoundly than SREBP-1a (Fig. 8 A), and the reverse was observed with respect to fatty acid synthesis (Fig. 8
B). As shown in Fig. 9, SREBP-1a raised the hepatic content
of triglycerides more than it did cholesterol, and therefore it
raised the triglyceride/cholesterol ratio (Fig. 9 C). On the other
hand, SREBP-2 raised the content of both lipids to approximately the same extent, and therefore the triglyceride/cholesterol ratio remained about the same as in the control animals(Fig. 9 C). Interestingly, although SREBP-1c was much less active than SREBP-1a or SREBP-2, it raised the triglyceride
content of the livers while leaving the cholesterol content unchanged. Thus SREBP-1c, like SREBP-1a, increased the triglyceride/cholesterol ratio.
The massive increase in cholesterol synthesis in the SREBP-2
transgenic mice (28-fold) correlates with the massive increasein the amount of mRNA for HMG CoA reductase (75-fold in
the line B mice). This was coupled with an 11-fold increase in
the amount of mRNA for HMG CoA synthase, which produces the substrate for the HMG CoA reductase reaction.
These results emphasize the importance of these two enzymes
as rate-controlling steps for the overall pathway of cholesterol
biosynthesis.
Considered together, the comparative results of Figs. 7–9
indicate that overexpression of dominant positive forms of
SREBP-1a and SREBP-1c favors triglyceride synthesis as opposed to cholesterol synthesis, whereas expression of SREBP-2
tends to favor the synthesis of cholesterol over triglyceride inlivers of transgenic mice.
The experiments with dominant positive transgenes are
conducted under highly artificial experimental conditions, yetthey have implications for normal physiology. The results suggest that SREBP-1a, -1c, and -2 may have subtly distinct rolesin the liver. Under normal conditions the mouse liver produces
relatively large amounts of SREBP-1c relative to SREBP-1a(12). The same is true of the human (12) and the hamster (14).
The function of the major SREBP-1c isoform may be to support fatty acid biosynthesis, and the smaller amounts of
SREBP-1a may support low rates of cholesterol biosynthesis
as well. On a low cholesterol chow diet, the mouse liver produces relatively little SREBP-2. The situation changes when
the liver is deprived of cholesterol by treatment with diets con-Figure 7. Relative levels of various mRNAs in livers of mice expressing transgenes encoding truncated SREBP-1c, -1a, and -2. Wild-type mRNAlevels in each experiment are arbitrarily set at 1.
The mRNA values for mice expressing truncated
SREBP-1c (15) and SREBP-1a (16) were measured by Northern gel analysis and are taken fromthe indicated publication. mRNA levels for thetransgenic SREBP-2 mice are from this paper. All
mice were fed the low carbohydrate/high protein diet for 2 wk. Transgenic SREBP-1a and
SREBP-2 mice were fasted for 4 h before killing.
Transgenic SREBP-1c mice were not fasted.
Transgenic Mice Expressing Truncated SREBP-2

2337

Figure 8. Rates of cholesterol and fatty acid synthesis in vivo in liversof mice expressing transgenes encoding truncated SREBP-1c, -1a,and -2. Values for SREBP-1a transgenic mice and their wild-type(WT) controls (mean6SEM for 4–6 mice) are reproduced from experiments described in Table III in reference 16. Synthesis rates for
SREBP-1c animals (7 mo of age) represent the mean6SEM of values
from three female and one male transgenic SREBP-1c mice as compared with four sex-matched littermate wild-type mice. Synthesis
rates for transgenic SREBP-2, line B mice (mean6SEM for fivemice) are derived from the animals described in Table II. All mice
were fed the low carbohydrate/high protein diet for 2 wk before theexperiment. The SREBP-1a and -2 mice and their wild-type controls
were fasted 2 and 4 h, respectively, before killing. The SREBP-1c
mice were studied in the nonfasted state. All mice were injected with
50 mCi of [3H]water intraperitoneally, and 1 h later the liver was removed for measurement of its content of 3H-labeled digitonin-precipitable sterols and fatty acids as described in Methods.

taining an HMG CoA reductase inhibitor (lovastatin) and abile acid–binding resin (Colestipol). Under these conditions,livers of hamsters (25) and mice (Shimano, H., J. Horton, J.L.
Goldstein, and M.S. Brown, unpublished observations) downregulate the production of the SREBP-1 isoforms and they upregulate production of SREBP-2. The proteolytic cleavage of
the SREBP-2 precursor is also enhanced, leading to a marked
increase in the content of the NH2-terminal segment of
SREBP-2 in liver nuclei (reference 25 and Shimano, H., J.
Horton, J.L. Goldstein, and M.S. Brown, unpublished observations). The current data suggest that the increase in SREBP-2,
at the expense of the SREBP-1 isoforms, would lead to a selective increase in cholesterol biosynthesis as opposed to fatty
acid biosynthesis. This would be an appropriate response tocholesterol deficiency.
2338

Horton et al.

Figure 9. Comparison of cholesterol and triglyceride content in liversof mice expressing transgenes encoding truncated SREBP-1c, -1a,and -2. Values for the transgenic SREBP-1c mice and their wild-type(WT) controls (mean6SEM for eight to nine mice) are taken fromdata of Table I in reference 15. Values for the transgenic SREBP-1a
mice and their WT controls (mean6SEM for six mice) are takenfrom data of Table I, Exp. B in reference 16. Values for the transgenic
SREBP-2, line B mice are from this paper. All mice were fed the
low carbohydrate/high protein diet for 2 wk before the experiment.
Transgenic SREBP-1a and SREBP-2 mice were fasted for 4 h before
killing. Transgenic SREBP-1c mice were not fasted.

The selective effect of SREBP-2 in stimulating cholesterolsynthesis was particularly apparent in adipose tissue. Here the
SREBP-2 transgenic mice showed a distinct increase in mRNAs
encoding the LDL receptor and cholesterol biosynthetic enzymes yet no change in the mRNAs of the fatty acid pathway(Fig. 4). It seems likely that the potential stimulating effects of
SREBP-2 on fatty acid-related genes is counterbalanced byother regulatory proteins that tend to limit fatty acid biosynthesis.
In addition to the differential effects of cholesterol andfatty acid biosynthesis, it is also possible that the three SREBP
isoforms may have different effects on transcription of othergenes whose existence is as yet unknown. Further elucidationof the effects of SREBPs on gene transcription will require detailed study of other genes that may be activated in the liversof the transgenic animals, as well as a more detailed comparison of the transcription-stimulating effects of the three iso-forms in tissue culture cells where a larger spectrum of quantitative measurements can be made. Studies along these lines
are now underway.

Acknowledgments
We thank Yuri Bashmakov, Scott Clark, Shan Maika, and Robin
Craddock for excellent technical assistance; Richard Gibson andLaura Quinlivan for invaluable help with the animals; and Dr. Laura
Woollett for assistance with the [3H]water experiments.
This work was supported by grants from the National Institutes of
Health (HL-20948), the Moss Heart Foundation, and the Perot Family Foundation. J.D. Horton is the recipient of a Postdoctoral Fellowship for Physicians from the Howard Hughes Medical Institute (Dallas, TX). I. Shimomura is the recipient of a Research Fellowship from
the Manpei Suzuki Diabetes Foundation of Tokyo, Japan.

References
1. Dietschy, J.M. 1997. Overview of cholesterol and lipoprotein metabolism
in the brain, liver and extrahepatic organs. Nutr. Metab. Cardiovasc. Dis. 7:162–168.
2. Hillgartner, F.B., L.M. Salati, and A.G. Goodridge. 1995. Physiologicaland molecular mechanisms involved in nutritional regulation of fatty acid synthesis. Physiol. Rev. 75:47–76.
3. Brown, M.S., and J.L. Goldstein. 1997. The SREBP pathway: Regulationof cholesterol metabolism by proteolysis of a membrane-bound transcription
factor. Cell. 89:331–340.
4. Kim, J.B., G.D. Spotts, Y.-D. Halvorsen, H.-M. Shih, T. Ellenberger,
H.C. Towle, and B.M. Spiegelman. 1995. Dual DNA binding specificity of
ADD1/SREBP1 controlled by a single amino acid in the basic helix-loop-helix
domain. Mol. Cell. Biol. 15:2582–2588.
5. Yokoyama, C., X. Wang, M.R. Briggs, A. Admon, J. Wu, X. Hua, J.L.
Goldstein, and M.S. Brown. 1993. SREBP-1, a basic helix-loop-helix leucine
zipper protein that controls transcription of the LDL receptor gene. Cell. 75:
187–197.
6. Hua, X., C. Yokoyama, J. Wu, M.R. Briggs, M.S. Brown, J.L. Goldstein,
and X. Wang. 1993. SREBP-2, a second basic-helix-loop-helix-leucine zipper
protein that stimulates transcription by binding to a sterol regulatory element.
Proc. Natl. Acad. Sci. USA. 90:11603–11607.
7. Ericsson, J., S.M. Jackson, B.C. Lee, and P.A. Edwards. 1996. Sterol regulatory element binding protein binds to a cis element in the promoter of thefarnesyl diphosphate synthase gene. Proc. Natl. Acad. Sci. USA. 93:945–950.
8. Guan, G., P.-H. Dai, T.F. Osborne, J.B. Kim, and I. Shechter. 1997. Multiple sequence elements are involved in the transcriptional regulation of the human squalene synthase gene. J. Biol. Chem. 272:10295–10302.
9. Kim, J.B., and B.M. Spiegelman. 1996. ADD1/SREBP1 promotes adipocyte differentiation and gene expression linked to fatty acid metabolism. Genes& Dev. 10:1096–1107.
10. Lopez, J.M., M.K. Bennett, H.B. Sanchez, J.M. Rosenfeld, and T.F. Osborne. 1996. Sterol regulation of acetyl CoA carboxylase: A mechanism for co-ordinate control of cellular lipid. Proc. Natl. Acad. Sci. USA. 93:1049–1053.
11. Ericsson, J., S.M. Jackson, J.B. Kim, B.M. Spiegelman, and P.A. Edwards. 1997. Identification of glycerol-3-phosphate acyltransferase as an adipocyte determination and differentiation factor 1 and sterol regulatory elementbinding protein-responsive gene. J. Biol. Chem. 272:7298–7305.
12. Shimomura, I., H. Shimano, J.D. Horton, J.L. Goldstein, and M.S.
Brown. 1997. Differential expression of exons 1a and 1c in mRNAs for sterol
regulatory element binding protein-1 in human and mouse organs and cultured
cells. J. Clin. Invest. 99:838–845.
13. Tontonoz, P., J.B. Kim, R.A. Graves, and B.M. Spiegelman. 1993.
ADD1: a novel helix-loop-helix transcription factor associated with adipocyte
determination and differentiation. Mol. Cell. Biol. 13:4753–4759.
14. Shimomura, I., Y. Bashmakov, H. Shimano, J.D. Horton, J.L. Goldstein, and M.S. Brown. 1997. Cholesterol feeding reduces nuclear forms of sterol regulatory element binding proteins in hamster liver. Proc. Natl. Acad. Sci.
USA. 94:12354–12359.
15. Shimano, H., J.D. Horton, I. Shimomura, R.E. Hammer, M.S. Brown,
and J.L. Goldstein. 1997. Isoform 1c of sterol regulatory element binding protein is less active than isoform 1a in livers of transgenic mice and in cultured
cells. J. Clin. Invest. 99:846–854.
16. Shimano, H., J.D. Horton, R.E. Hammer, I. Shimomura, M.S. Brown,
and J.L. Goldstein. 1996. Overproduction of cholesterol and fatty acids causes
massive liver enlargement in transgenic mice expressing truncated SREBP-1a.
J. Clin. Invest. 98:1575–1584.
17. Hamilton, J.G., and K. Comai. 1988. Rapid separation of neutral lipids,
free fatty acids and polar lipids using prepacked silica Sep-pak columns. Lipids.
23:1146–1149.
18. Turley, S.D., M.W. Herndon, and J.M. Dietschy. 1994. Reevaluation
and application of the dual-isotope plasma ratio method for the measurementof intestinal cholesterol absorption in the hamster. J. Lipid Res. 35:328–339.
19. Short, M.K., D.E. Clouthier, I.M. Schaefer, R.E. Hammer, M.A. Magnuson, and E.G. Beale. 1992. Tissue-specific, developmental, hormonal, and dietary regulation of rat phosphoenolpyruvate carboxykinase-human growth hormone fusion genes in transgenic mice. Mol. Cell. Biol. 12:1007–1020.
20. Chomczynski, P., and N. Sacchi. 1987. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 162:156–159.
21. Shimano, H., I. Shimomura, R.E. Hammer, J. Herz, J.L. Goldstein, M.S.
Brown, and J.D. Horton. 1997. Elevated levels of SREBP-2 and cholesterolsynthesis in livers of mice homozygous for a targeted disruption of the SREBP-1
gene. J. Clin. Invest. 100:2115–2124.
22. Zhu, Y., K. Alvares, Q. Huang, M.S. Rao, and J.K. Reddy. 1993. Cloning of a new member of the peroxisome proliferator activated receptor genefamily from mouse liver. J. Biol. Chem. 268:26817–26820.
23. Sato, R., J. Yang, X. Wang, M.J. Evans, Y.K. Ho, J.L. Goldstein, and
M.S. Brown. 1994. Assignment of the membrane attachment, DNA binding,and transcriptional activation domains of sterol regulatory element binding
protein-1 (SREBP-1). J. Biol. Chem. 269:17267–17273.
24. Sharp, D., L. Blinderman, K.A. Combs, B. Kienzle, B. Ricci, K. WagerSmith, C.M. Gil, C.W. Turck, M.-E. Bouma, D.J. Rader, et al. 1993. Cloning
and gene defects in microsomal triglyceride transfer protein associated with
abetalipoproteinaemia. Nature. 365:65–69.
25. Sheng, Z., H. Otani, M.S. Brown, and J.L. Goldstein. 1995. Independent
regulation of sterol regulatory element binding proteins 1 and 2 in hamster
liver. Proc. Natl. Acad. Sci. USA. 92:935–938.

Transgenic Mice Expressing Truncated SREBP-2

2339