Metabolism and Bioenergetics:

CMO1 Deficiency Abolishes Vitamin A

Production from -Carotene and Alters
Lipid Metabolism in Mice
Susanne Hessel, Anne Eichinger, Andrea
Isken, Jaume Amengual, Silke Hunzelmann,
Ulrich Hoeller, Volker Elste, Willi Hunziker,
Regina Goralczyk, Vitus Oberhauser,
Johannes von Lintig and Adrian Wyss

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J. Biol. Chem. 2007, 282:33553-33561.

doi: 10.1074/jbc.M706763200 originally published online September 12, 2007

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 46, pp. 3355333561, November 16, 2007
2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

CMO1 Deficiency Abolishes Vitamin A Production from

-Carotene and Alters Lipid Metabolism in Mice*
Received for publication, August 14, 2007, and in revised form, September 12, 2007 Published, JBC Papers in Press, September 12, 2007, DOI 10.1074/jbc.M706763200

Susanne Hessel1, Anne Eichinger1, Andrea Isken, Jaume Amengual2, Silke Hunzelmann, Ulrich Hoeller,
Volker Elste, Willi Hunziker, Regina Goralczyk, Vitus Oberhauser, Johannes von Lintig3, and Adrian Wyss4
From the Institute of Biology I, Animal Physiology and Neurobiology, Hauptstrasse 1, D-79104 Freiburg, Germany,

DSM Nutritional Products Ltd., R & D Human Nutrition and Health, P.O. Box 3255, CH-4002 Basel, Switzerland,
and Frimorfo SA, Chemin du Musee, CH-1700 Fribourg, Switzerland

Dietary lipids are precursors for signaling molecules that

control many facets in cell physiology. As the classic example,

* This work was supported by a grant from the Ministry of Science and Art
Baden-Wurttemberg (to J. v. L.). The costs of publication of this article were
defrayed in part by 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.
1
These authors contributed equally to this work.
2
Supported by an exchange grant from the European Nutrigenomic
Organization.
3
To whom correspondence may be addressed: Dept. of Neurobiology and
Animal Physiology, Institute of Biology I, Hauptstrasse 1, Albert-Ludwig
University, 79104 Freiburg, Germany. Tel.: 49-761-203-2539; Fax: 49-761203-2921; E-mail: lintig@biologie.uni-freiburg.de.
4
To whom correspondence may be addressed: DSM Nutritional Products,
4303 Kaiseraugst, Switzerland. Tel.: 41-61-688-57-92; Fax: 41-61-688-1640; E-mail: adrian.wyss@dsm.com.

NOVEMBER 16, 2007 VOLUME 282 NUMBER 46

fat-soluble vitamin A (all-trans-retinol) is essential for processes ranging from development to vision and cell proliferation (13). Retinol is the precursor for at least two critical
metabolites, 11-cis-retinal, the chromophore of visual G-protein-coupled receptors (4), and retinoic acid (RA).5 Alltrans-RA and 9-cis-RA regulate gene expression via heterodimeric nuclear receptors consisting of an RA receptor and
a retinoid X receptor (RXR) (5, 6). Both are ligand-dependent
transcription factors belonging to the superfamily of nuclear
hormone receptors (7). Additionally, RXRs form heterodimers
with other members of the nuclear receptor family (8), including the peroxisome proliferator-activated receptors (PPARs).
Because animals, including humans, are unable to synthesize
vitamin A de novo, all retinoids (vitamin A and its derivatives)
derive from the oxidative cleavage of dietary provitamin A
carotenoids, mainly -carotene (9 11). How this conversion of
-carotene occurs (centric and/or eccentric cleavage) is still a
matter of debate (1214). Recently, two different carotenoidmonooxygenases, CMO1 and CMO2, were molecularly identified in animals, including humans (15). Both belong to a family
of structurally related nonheme iron oxygenases, common to
all taxa (16 18). Biochemical analyses revealed that CMO1 is a
carotenoid-15,15-oxygenase, which converts -carotene to
retinaldehyde by a centric oxidative cleavage at the C15,C15
double bond (19 24). From this cleavage product, all biologically active retinoids can be produced by endogenous metabolic
pathways, including 11-cis-retinal and RA. The CMO1 gene has
been shown to be transcriptionally regulated by the action of
PPARs and RXRs in both mice and humans (25, 26). This characteristic indicates a regulatory interlink between carotenoid
and fatty acid metabolism at this level. The second carotenoidmetabolizing enzyme, CMO2, catalyzes an eccentric oxidative
cleavage of carotenoids, such as -carotene and lycopene, at the
C9,C10 double bond (27, 28). The physiological role of this
carotenoid oxygenase is still poorly understood.
In most mammals, including humans, dietary provitamin A
carotenoids can maintain all vitamin A-dependent physiological functions. But it is still not clear whether dietary vitamin A
possess the full biological activity of the provitamin. There is
evidence in the literature that carotenoid oxygenases can tis-

The abbreviations used are: RA, retinoic acid; FFA, serum free fatty acid;
PPAR, peroxisome proliferator-activated receptor; RXR, retinoid X receptor; HPLC, high pressure liquid chromatography; qRT, quantitative real
time.

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Carotenoids are currently investigated regarding their potential to lower the risk of chronic disease and to combat vitamin A
deficiency in humans. These plant-derived compounds must be
cleaved and metabolically converted by intrinsic carotenoid
oxygenases to support the panoply of vitamin A-dependent
physiological processes. Two different carotenoid-cleaving
enzymes were identified in mammals, the classical carotenoid15,15-oxygenase (CMO1) and a putative carotenoid-9,10oxygenase (CMO2). To analyze the role of CMO1 in mammalian
physiology, here we disrupted the corresponding gene by targeted homologous recombination in mice. On a diet providing
-carotene as major vitamin A precursor, vitamin A levels fell
dramatically in several tissues examined. Instead, this mouse
mutant accumulated the provitamin in large quantities (e.g. as
seen by an orange coloring of adipose tissues). Besides impairments in -carotene metabolism, CMO1 deficiency more generally interfered with lipid homeostasis. Even on a vitamin
A-sufficient chow, CMO1/ mice developed a fatty liver and
displayed altered serum lipid levels with elevated serum unesterified fatty acids. Additionally, this mouse mutant was more
susceptible to high fat diet-induced impairments in fatty acid
metabolism. Quantitative reverse transcription-PCR analysis
revealed that the expression of peroxisome proliferator-activated receptor -regulated marker genes related to adipogenesis was elevated in visceral adipose tissues. Thus, our study identifies CMO1 as the key enzyme for vitamin A production and
provides evidence for a role of carotenoids as more general regulators of lipid metabolism.

CMO1 Deficiency Impairs Lipid Metabolism

sue-specifically impact physiological processes. In Drosophila,
mutations in CMO1 are associated with blindness (29). In
zebrafish, CMO1 is required for RA production in certain
developing cells (30). Moreover, evidence for mammals exists
that carotenoid derivatives produced by eccentric cleavage of
-carotene, such as -14-apocarotenal, can influence gene regulation via PPARs and RXRs (31). In humans, mutations in
carotenoid monooxygenases have not yet been described in
association with any known physiological disorder. Therefore,
to learn more about mammalian carotenoid metabolism and
function, here we have established and characterized a mouse
model with a disruption of the CMO1 gene.

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EXPERIMENTAL PROCEDURES
Targeting Construct, Electroporation of Embryonic Stem
Cells, and Generation of CMO1/ ChimeraTo obtain a
homozygous CMO1 knock-out mouse, we used homologous
recombination in TC1 ES cells to replace exon 2 and exon 3 of
the wild-type CMO1 gene by an IRES-lacZ gene and a neomycinR cassette (Fig. 1). Three clones were isolated by PCR screening from a 129SvJ mouse BAC library (Genome Systems, St.
Louis, MO). They span three exons and a genomic region of
19.5 kb of the CMO1 gene. The flanking sequences (5 arm,
3.9-kb SacI-ApaI fragment; 3 arm, 3.4-kb ApaI-ApaI) were
cloned into the pGL-1 vector. Approximately 8.8 kb, including
half of exon 2 and exon 3, were deleted from the wild-type
CMO1 allele and replaced by an IRES-lacZ cassette, followed by
a neoR cassette between two loxP sites (Fig. 1). TC1 ES cells
(embryonic stem cells from mouse strain 129SvJ/SvEvTac)
were routinely cultured on mitomycin C-treated embryonic
fibroblasts, in a humidified atmosphere at 37 C, 10% CO2 and
subjected to G418 selection. For the electroporation into TC1
ES cells, 0.8 ml of a single cell suspension (5 106 cells/ml of
phosphate-buffered saline without Ca2 and Mg2) were
placed into a Bio-Rad electroporation cuvette. After the
addition of 20 g of the purified, NotI-linearized targeting
construct, the electroporation was performed in a Bio-Rad
GenePulser (Bio-Rad) at 240 V, 500 microfarads. After electroporation, the cells were seeded onto neomycin-resistant
embryonic fibroblasts. The selection with 200 mg/ml G418 was
started after 48 h. Individual clones were isolated 10 12 days
after electroporation. All of the clones were screened for
homologous recombination by Southern blotting using two
specific probes. Two clones that have undergone homologous
recombination were selected and injected into blastocytes, and
approximately 200 blastocysts were transferred into pseudopregnant foster mothers. Chimerism was estimated according
to the coat color of the litter. The male chimeric mice were
backcrossed with female C57BL/6 to produce heterozygous
and subsequently homozygous CMO1/ mice.
Confirmation of the Genotype by Southern Blot and PCR
The Easy-DNA kit (Invitrogen) was used to isolate genomic
DNA from mouse tail biopsies. Genotyping was carried out
both by Southern blot analysis and by a combination of different PCRs (Fig. 1). PCR was performed with the following primers: neomycin up, 5-ATG ATT GAA CAA GAT GGA TTG
CAC GCA G-3; neomycin down, 5-GAT ATT CGG CAA
GCA GGC ATC GCC AT-3; Cmo1up, 5-GAG AGA GCA

AGT ACA ACC ATT GGT TTG ATG-3; Cmo1down, 5-CTT

GTA AGA CTG TAA ATC CTG GTT TGA GAC-3; LacZ
long forward primer (5-CCT CAA TTT GCT TTT TGG TCA
TGG TG-3) and reverse primer (5-CTC GCC GCA CAT
CTG AAC TTC AG-3); neomycin long forward primer (5AGG TCT AAT TCC ATC AGA AGC-3) and reverse primer
(5-TCA GCC ACT TCT CTG TGT CGA G-3).
For Southern blot analysis, 20 g of genomic DNA were
digested with KpnI (3 arm) or EcoRV (5 arm). After electrophoretic separation, DNA was transferred onto a Zeta probe
membrane (Bio-Rad), and hybridization was carried out overnight at 65 C in ExpressHyb hybridization solution (Clontech)
using [-32P]dCTP-labeled 3 and 5 probes. The membrane
was washed twice with 2 SSC and once with 0.1 SSC, 0.1%
SDS at room temperature for 20 min each.
Animals, Diets, and Experimental ProceduresIn all experiments, we used C57/BL6;129SvJ-CMO1tm1dnp (CMO1/)
mice. For the analysis of vitamin A and lipid metabolism under
-carotene supplementation (-carotene feeding study), we
used mixed background F1 generation of C57/BL6;129SvJ mice
(RCC Ltd.) as wild-type controls. For the analysis of vitamin A
and lipid metabolism on the vitamin A-sufficient chow, we used
C57/BL6 (Charles River) mice as wild-type controls. Animal
experiments were performed in accordance with Swiss and
German animal protection laws by the guidelines of the local
veterinary authorities. Mice were maintained under environmentally controlled conditions (temperature 24 C, 12 h/12 h
light/dark cycle) and had ad libitum access to feed and water in
all experiments. For the -carotene supplementation study,
basic feed consisted of the powdered SsniffEF 1/51 diet (Ssniff
GmbH, Soest, Germany), containing a residual amount of 0.15
IU/g vitamin A. In the -carotene feeding study, this chow was
supplemented with -carotene beadlets (10% CWS; DSM Ltd.,
Sisseln, Switzerland). The final amount of -carotene in the
feed was 1 mg/g. In all other experiments, mice were routinely
fed with KLIBA 3430 chow (Provimi Kliba AG, Kaiseraugst,
Switzerland) containing 14 IU/g vitamin A. HPLC analysis
revealed that this chow contains the carotenoids: -carotene,
zeaxanthin, and lutein. The high fat chow consisted of the
KLIBA 3430 enriched with 30% (w/w) soybean oil and was purchased from Provimi Kliba AG (Kaiseraugst, Switzerland). For
plasma and serum preparations, first blood was taken from the
vena cava under deep anesthesia. The animals were then killed
prior to organ removal.
Immunoblot AnalysisFor determination of blood RBP4
levels, a commercially available polyclonal antiserum raised
against human RBP4 (Dako Cytomation) was used in a dilution
of 1:1000. For the tubulin loading control, we used the antiserum AB6046 (Abcam, Cambridge, UK) in a dilution of 1:500.
For determination of CMO1, we used an antiserum previously
described (30). Secondary antibodies were horseradish peroxidase-conjugated anti-rabbit and anti-mouse IgG whole molecules (Sigma). Immunoblots were developed with the ECL
system (Amersham Biosciences). Quantification of bands
was performed by the Quantity-one (version 4.6) software
(Bio-Rad).
HPLC Separation for Retinoids and CarotenoidsRetinoids
and carotenoids were extracted from tissues and plasma under

CMO1 Deficiency Impairs Lipid Metabolism

NOVEMBER 16, 2007 VOLUME 282 NUMBER 46

GAT AAA GCC ATT-3; ACOX1, 5-GAT TGG TAG AAA

TTG CTG CAA AAA-3 and 5- ACG CCA CTT CCT TGC
TCT TC-3; SREBP1c, 5-CCA GAG GGT GAG CCT GAC
AA-3 and 5-AGC CTC TGC AAT TTC CAG ATC T-3;
FASN, 5-CCA AGC AGG CAC ACA CAA-3 and 5-CAC
TCA CAC CCA CCC AGA-3; SCD1, 5-TTC CGC CAC TCG
CCT ACA-3 and 5-CTT TCC CAG TGC TGA GAT CGA-3;
CD36, 5-AAT TAG AAC CGG GCC ACG TA-3 and 5-CGC
CAA CTC CCA GGT ACA A-3; FABP4, 5-ATG TGT GAT
GCC TTT GTG G-3 and 5-CTG TCG TCT GCG GTG ATT
T-3. As housekeeping gene, we used -actin (5-ACG GGC
ATT GTG ATG GAC TC-3 and 5-GTG GTG GTG AAG
CTG TAG CC-3). qRT-PCR was performed in a total volume
of 25 l, consisting of diluted (1:50) cDNA template, forward
and reverse primers (12 M each), and SYBR Green ROX Mastermix (ABgene) with an ABI prism 7000 (Applied Biosystems,
Germany). After an initial enzyme activation (95 C for 15 min),
40 cycles at 60 C were performed for anneal/extension steps,
and fluorescence was measured. In order to verify purity of PCR
products, we performed 2% agarose gel electrophoresis. Additionally, a dissociation curve program was performed after each
reaction. Relative quantification of target genes was calculated
according to Ref. 34, based on the efficiency of each reaction
and the crossing point deviation of each sample versus a control
and expressed in comparison with a reference gene (-actin).
Statistical AnalysisThe data are presented as means S.D.
Students t test was used to analyze data between the controls
and knock-out strains. p values of 0.05 were considered
significant.

RESULTS
Mouse CMO1 Gene and Targeting ConstructThe CMO1
gene appears to be present as a single copy in the mouse (and
human) genome. The entire mouse CMO1 gene sequence can
be found at the NCBI site on the World Wide Web as a complement (44443023.44480639) at locus NT_078575 (GenBankTM) containing 37,617 bp. CMO1 is expressed in multiple
tissues, such as the intestine, testis, liver, spleen, and adipocytes
(21, 22, 25).
A mouse CMO1 genomic sequence was isolated by screening
of a 129SvJ mouse BAC library. A targeting vector was constructed to replace half of exon 2 and exon 3 with an IRES-lacZ
cassette and a neomycinR cassette (Fig. 1A), linearized with NotI
and electroporated into ES cells (129S6/SvEvTac). ES cells were
injected into C57BL/6 blastocysts. Initial screening for homologous recombination was carried out by Southern blot analysis
(Fig. 1C). The male chimeric mice were backcrossed with
female C57BL/6 mice to produce heterozygous and subsequently homozygous CMO1/ mice. The wild-type allele was
routinely identified using the primer pair CMO1up and
CMO1down (diagnostic fragment 351 bp) and the knock-out
gene using CMO1up and lacZlongdown (diagnostic fragment
588 bp) (Fig. 1B). The expression of CMO1 was abolished in the
liver of CMO1/ mice as determined by immunoblotting (Fig.
1D). CMO1/ mice developed normally, and females and
males were fertile on a standard mouse chow, thus excluding a
developmental role of CMO1 and -carotene when preformed
vitamin A is available.
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dim red safety light (600 nm). Briefly, tissues (20 40 mg) were
homogenized in 200 l of 2 M hydroxylamine (pH 6.8) and 200
l of methanol with a glass homogenizer. For determination of
-carotene and vitamin A blood levels, 200 l of plasma was
added to 200 l of methanol. Then 400 l of acetone was added
either to plasma or tissue extracts. Extraction of carotenoids
and retinoids was performed with petrolether. The extraction
was repeated three times, and the collected organic phases were
dried under a stream of nitrogen and redissolved in HPLC solvent. HPLC separation of carotenoids and retinoids and quantification of peak integrals was performed as previously
described (19). Solvents for HPLC and extraction were purchased in HPLC grade from Merck.
Determination of Tissue and Serum Lipids and Glucose Tolerance TestDetermination of the levels of free fatty acids, triglycerides, cholesterol esters, and glutamate pyruvate transaminase in serum and tissues was carried out by the Central
Laboratory Unit of the University Hospital Freiburg (Germany). For determination of the total lipid content, liver samples (515 mg) were homogenized in phosphate-buffered saline
(pH 7.5). Total lipid content was determined by Merckotest
3321 Total Lipids (Diagnostica Merck) according to the manufacturers protocol. Glucose tolerance tests were performed by
intraperitoneal injection of glucose (1 g/kg body weight) after
overnight starvation. Blood glucose levels were determined by a
glucometer (Accu-Chek Aviva; Roche Applied Science) according to the manufacturers protocol. For this purpose, tail tips of
the mice were nicked with a fresh razor blade for the collection
of blood samples.
HistologyFor paraffin sections, livers were fixed in neutral
buffered formalin and processed routinely into paraffin blocks.
The embedded tissues were cut into 6-m slices, mounted on
charged adhesive slides, and dried overnight at 50 C. Slides
were then deparaffinized in xylene and rehydrated in graded
alcohols to distilled water. Representative histologic sections of
each specimen were stained with hematoxylin/eosin (Merck,
Germany) according to the standard staining method of Ref. 32
or by Massons trichrome stain (Sigma) according to the manufacturers instructions. For cryosections, livers were cryoprotected in 30% sucrose overnight. The tissues were embedded in
1.5% agarose and sectioned with a cryostat (Microm HM
500-O) into 12-m slices. After fixation, the sections were
stained with Sudan Red 7B (Sigma) according to Refs. 32 and 33
for general histological stain methods.
RNA Preparation and Quantitative Real Time PCR
(qRT-PCR)Total RNA was isolated from liver and visceral
adipose tissues with TRIzol reagent (Invitrogen) according to
the manufacturers protocol. RNA samples were cleaned up,
DNase I-digested with the RNeasy minikit (Qiagen), and quantified spectrophotometrically. Integrity of the extracted RNA
was verified by gel electrophoresis. For cDNA synthesis, 0.5 g
of total RNA (in a final volume of 25 l) was denatured at 65 C
for 10 min. Then cDNA synthesis was carried out for 15 min at
20 C and 45 min at 42 C with reverse transcriptase (MuLV RT;
Applied Biosystems). The reaction was stopped at 95 C for 5
min using a thermal cycler (Progene). For qRT-PCR analysis of
target genes, the following primers were used: PPAR, 5-CGA
TGC TGT CCT CCT TGA TGA-3 and 5-CTC GCG TGT

CMO1 Deficiency Impairs Lipid Metabolism

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control and CMO1/ offspring.

Upon weaning, animals were continuously raised on a diet low in
vitamin A, but they additionally
received -carotene (1000 ppm).
After 16 weeks, the consequence of
CMO1 deficiency was already visible upon sectioning. CMO1/
mice but not wild-type control animals showed an orange coloring in
visceral adipose tissue (Fig. 2, A and
B), in subcutaneous adipose tissue,
and in the intestine (Fig. 2, C and D).
Retinoid production from -carotene already takes place in intestinal cells. Therefore, we first performed HPLC analysis with
intestinal lipid extracts. This analysis confirmed -carotene accumulation in CMO1/ mice, whereas
/
FIGURE 1. Targeting strategy and creation of CMO1
mice. A, schematic map for the recombinant and the in controls besides -carotene (Fig.
wild type (wt) CMO1 alleles. The arrows indicate the positions of the PCR primers used for genotyping. In the 2F), cleavage products, such as retirecombinant allele, half of exon 2 and exon 3 were replaced by an IRES-LacZ and a neomycin cassette.
B, multiplex PCR showing the wild-type genotype, the homozygous CMO1/ genotype, and the heterozy- nyl esters and all-trans-retinol, were
gous CMO1/ genotype. C, Southern blot analysis of genomic DNA restricted with KpnI and hybridized with present (Fig. 2, G and H). This lack
the 3 probe isolated from wild-type (lane 1), CMO1/ (lane 2), and CMO1/ (lane 3) mice. D, immunoblot of intestinal -carotene conversion
analysis with protein extracts (100 g/lane) isolated from livers of wild type and homozygous CMO1/ mice
/
mice was accompaconfirms the absence of the CMO1 protein (top). Bottom, tubulin loading control. Primer pairs used for geno- in CMO1
typing and the positions of probes for Southern blot analysis are indicated. ex 1, exon 1; ex 2, exon 2; ex 3, exon nied by a 35-fold increase of
3 of the CMO1 wild-type allele. neoR, neomycin resistance; lacZ, -galactosidase.
-carotene blood levels (Fig. 2E). To
further assess the consequence of
CMO1 deficiency, we determined -carotene and retinoid
(retinyl esters and retinol) levels in additional tissues that are
known to express the CMO1 gene (Fig. 3). In all of these tissues,
levels of -carotene were increased as compared with control
animals. Conversely, tissue vitamin A levels were decreased in
CMO1/ mice as compared with controls. Only in the kidney
were retinol levels low and indistinguishable between
CMO1/ and control animals. Thus, CMO1 deficiency
impaired -carotene metabolism, and the provitamin accumulated in several tissues.
CMO1/ Mice Develop Liver Steatosis when -Carotene Is
Provided as the Major Dietary Source for Vitamin AWe next
FIGURE 2. -Carotene and retinoid levels in mice upon -carotene sup- looked to see whether CMO1 deficiency is associated with any
plementation. Shown are close-ups of the internal organs of the wild-type
additional phenotype under -carotene supplementation. We
control mouse (A) and the CMO1/ mouse (B). C, view of the subcutaneous
/
mice had a pale color. Determiadipose tissue in control (left) and CMO1/ (right) mice. D, isolated and found that livers of CMO1
/
washed intestines of control (left) and CMO1
(right) mice. E, -carotene nation of the lipid content of the livers revealed a significant
plasma levels; F, -carotene levels in the intestine; G, retinyl ester levels in the
intestine; H, all-trans-retinol levels in the intestine. wt, wild-type control; CAR, increase over control levels (Fig. 4C). Histological analysis of
-carotene; RE, retinyl esters; ROL, all-trans-retinol. Plasma -carotene levels liver slices showed abnormalities of hepatocytes in centrolobuare the mean S.D. of five animals/genotype. Levels of -carotene and reti- lar and midzonal regions (Fig. 4, D and E). These hepatocytes
noids of the intestine give the mean S.D. of three animals/genotype.
contained large droplets positively staining for lipids (Fig. 4, A
and B). HPLC analysis revealed that liver vitamin A (retinyl
-Carotene Metabolism Is Impaired in CMO1 Deficiency esters and retinol) was significantly lower as compared with
To analyze the consequences of CMO1 deficiency on a diet that controls (Fig. 4G). Instead, -carotene accumulation was found
provides -carotene as the major dietary source for vitamin A, in the livers of CMO1/ mice (Fig. 4H). This impairment in
we performed a feeding experiment. To deplete endogenous liver vitamin A homeostasis was also seen by an accumulation
stores for retinoids (i.e. in the liver), we already subjected con- of the serum retinol-binding protein (RBP4) (Fig. 4, F and I).
trol and CMO1/ breeders to a diet poor in vitamin A (0.15 RBP4 is produced in the liver and secreted in a vitamin A-deIU/g). This dietary regimen caused no obvious clinical symp- pendent manner to deliver vitamin A to peripheral target tistoms related to vitamin A deficiency in breeders or likewise in sues (35). Thus, CMO1/ mice developed a fatty liver and

CMO1 Deficiency Impairs Lipid Metabolism

displayed depleted vitamin A liver stores when -carotene was

provided as the major dietary source for vitamin A.
Liver Steatosis Is Related to CMO1 Deficiency and Develops
Independently of the Vitamin A Status of the Diet-Carotene
derivatives, such as RA, can influence liver fatty acid metabolism (e.g. genetic disruption of RA signaling can cause liver steatosis and steatohepatitis) (36). We found that CMO1/ mice
developed a fatty liver when -carotene was provided as the
major precursor for vitamin A. Therefore, we next wondered
whether this liver phenotype can be prevented by feeding mice
with preformed vitamin A. To address this question, we bred
and raised mice on a vitamin A-sufficient standard chow (14 IU
of vitamin A/g). In a first set of experiments, we performed
Massons trichrome staining for collagen of liver sections of
CMO1-deficient animals with different ages (n 2 per age)
(Fig. 5, AC). We found that hepatocytes of 3-month-old animals already contained some lipid droplets (Fig. 5B). Droplets
were larger and more widespread in livers of 12-month-old animals (Fig. 5C), but this analysis revealed that this alteration was
not accompanied by enhanced collagen deposition, a characteristic of liver fibrosis (37).
For a developmental and more defined analysis, we used
25-week-old CMO1/ mice and age-matched controls (n
10/genotype), raised and bred under the same environmental
and dietary conditions. CMO1/ mice had significantly eleNOVEMBER 16, 2007 VOLUME 282 NUMBER 46

FIGURE 5. Liver phenotype of CMO1/ mice raised on the vitamin A-sufficient standard chow. AC, representative liver sections of CMO1/ mice
stained with Massons trichrome. Shown are 1-month-old (A), 3-month-old
(B), and 12-month-old (C) CMO1/ mice. D, total liver lipid content of
25-week-old mice (values are means S.D.; n 8; *, p 0.02). E, liver triglycerides in 25-week-old mice (values are means S.D.; n 8; *, p 0.02).
F, serum all-trans-retinol levels in female and male mice. Values are means
S.D. (n 5/genotype and gender; *, p 0.02). Scale bar, 100 m. TG, triglycerides; ROL, all-trans-retinol; wt, wild type.

vated levels of total lipids as compared with control animals

(Fig. 5D). Additionally, livers of CMO1/ mice contained significantly more triglycerides (Fig. 5E). Histology of liver slices
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FIGURE 3. -carotene and retinoid levels in tissues of CMO1/ and control mice upon -carotene supplementation. Top, total vitamin A levels
(retinyl esters and all-trans-retinol) in selected tissues. Bottom, -carotene
levels in selected tissues. Value gives the mean S.D. (n 3/genotype for
lung and kidney; n 2/genotype for testis and uterus). CAR, -carotene; n.d.,
not detectable; wt, wild type.

FIGURE 4. CMO1/ mice develop liver steatosis and have depleted vitamin A stores in the liver upon -carotene supplementation. A and B, cryosections of livers stained with oil red. A, control mouse; B, CMO1/ mouse.
C, total liver lipid content of livers of CMO1/ and control mice. Values are
means S.D. (n 4/genotype; *, p 0.04; Students t test). D and E, representative liver sections of CMO1/ and control animals upon hematoxylin/
eosin staining. D, control mouse; E, CMO1/ mouse. F, immunoblot analysis
for RBP4 of liver protein extracts from control and CMO1/ mice supplemented with -carotene (CAR) or raised on the vitamin A-sufficient (vit A)
standard chow. G, total vitamin A levels (retinyl esters and all-trans-retinol) in
the liver. H, -carotene levels in the liver. Values in G and H are means S.D.
(n 5/genotype). I, densitometric quantification of RBP4 levels in immunoblot analysis. The values represent the results of three animals/genotype and
dietary regimen. wt, wild-type control animals; scale bars, 100 m; wt, wild type.

CMO1 Deficiency Impairs Lipid Metabolism

33558 JOURNAL OF BIOLOGICAL CHEMISTRY

FIGURE 6. Levels of CMO1 and CMO2 mRNA and levels of marker genes
of fatty acid metabolism. A, CMO1 and CMO2 mRNA levels determined by
real time PCR analysis. B, relative mRNA levels of marker genes of lipid
metabolism in livers determined by qRT-PCR. Values are means S.D.; n
9/genotype; *, p 0.01; **, p 0.001. C, relative mRNA levels of marker
genes of PPAR-regulated target genes in visceral adipose tissue determined
by qRT-PCR. Values are means S.D.; n 5/genotype; *, p 0.01. AU, arbitrary units; vAT, visceral adipose tissue; ACOX1, acyl-CoA-oxidase 1; CD36,
scavenger receptor CD36; FABP4, fatty acid-binding protein 4; FASN, fatty acid
synthase; SCD1, steaoryl-CoA-desaturase 1, SREBP1c, sterol regulatory element-binding protein 1c; wt, wild type.

for liver injury (41), were significantly higher in the CMO1deficient mutant (Fig. 7E). More interestingly, levels of unesterified serum free fatty acids (FFAs) were significantly elevated
in CMO1/ mice (Fig. 7F). Since the rate of hepatic uptake of
FFA is unregulated and is directly proportional to its serum
concentrations (42), this characteristic may explain the liver
phenotype in this mouse mutant.
Since CMO1/ mice showed alterations in fatty acid
metabolism, we finally asked whether CMO1/ mice are more
susceptible to diet-induced obesity. To approach this question,
we used 20-week-old female CMO1/ and control mice (n
5/genotype). The average weight of these mice was comparable
at the beginning of the study. They were then fed with the
standard chow supplemented with 30% fat in the form of soybean oil. We monitored weight gain weekly during a period of 8
weeks. CMO1/ mice gained significantly more weight as
compared with control animals (Fig. 7G). Analysis of the total
lipid content of livers showed that it increased in both genoVOLUME 282 NUMBER 46 NOVEMBER 16, 2007

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again revealed lipid droplets in hepatocyctes in CMO1/ mice

but not in control animals (data not shown). Thus, CMO1/
mice developed liver steatosis on a vitamin A-sufficient chow.
To exclude the possibility that CMO1 deficiency interferes
with liver vitamin A homeostasis even when preformed vitamin
A is available with the diet, we performed HPLC analysis to
measure retinoid levels. Liver vitamin A (retinyl esters and alltrans-retinol) was in the same range in CMO1/ and control
animals (3 mol 0.2 g1 in both genotypes). Additionally,
levels of RBP4 in liver protein extracts were comparable in both
genotypes (Fig. 4, F and I). We also determined the levels of
circulating retinol and -carotene in the blood. In CMO1/
mice, we found low but significant -carotene serum levels
(0.02 0.005 M), whereas -carotene was below detection
levels in control animals. Interestingly, serum all-trans-retinol
levels were slightly but significantly reduced in both female and
male CMO1/ mice as compared with controls (Fig. 5F).
PPAR Signaling Is Altered in Adipocytes of CMO1/ Mice
CMO1 has been identified as a PPAR- and RXR-responsive target gene in mice (25). These lipid-activated nuclear receptors
play an essential role in regulating fatty acid metabolism (38).
-Carotene-derived retinaldehyde has been recently shown to
protect against diet-induced obesity by inhibiting PPAR activity (39). Retinaldehyde is also the precursor for RA and 9-cisRA, which activate RA receptors and RXRs. Furthermore, putative CMO2-derived -carotene cleavage products can inhibit
PPAR activities as well as RXR activities in cell culture (31). We
found CMO2 mRNA expression in the liver and visceral adipose tissue of both CMO1/ and control animals (Fig. 6A).
Therefore, we now wondered whether the expression of genes
involved in fatty acid metabolism is altered in CMO1 deficiency. To address this question, we performed qRT-PCR analysis for genes of both fatty acid catabolism and synthesis (Fig.
6B). In the liver, mRNA levels of PPAR were comparable
between genotypes. Fatty acid oxidase 1 (ACOX1) mRNA levels
were significantly elevated in CMO1/ mice. Fatty acid synthase (FASN) and SREBP1c mRNA levels were within the same
range in the different genotypes. In contrast, levels of steaorylCoA-desaturase (SCD1) mRNA were significantly decreased in
livers of CMO1/ mice. We next analyzed the mRNA levels of
PPAR target genes in visceral adipose tissue (Fig. 6C). This
analysis revealed that mRNA levels of the fatty acid-binding
protein 4 (FABP4) and the scavenger receptor CD36 were significantly elevated in CMO1/ mice. Thus, CMO1/ mice
show altered mRNA expression of genes involved in fatty acid
metabolism in the liver and increased mRNA levels of PPARactivated genes in visceral adipose tissues.
CMO1/ Mice Display Elevated Levels of Serum Free Fatty
Acids and Are More Susceptible to Diet-induced ObesityNonalcoholic liver steatosis, as we here found in CMO1/ mice, is
related to obesity and/or type 2 diabetes (40). Therefore, we
analyzed relevant serum parameters of energy metabolism.
Serum glucose levels were comparable between the genotypes
(Fig. 7A). Glucose tolerance assays with 28-week-old animals
also revealed no significant differences in glucose clearance
(Fig. 7B). Serum cholesterol ester and triglyceride levels also
showed no differences between genotypes (Fig. 5, C and D).
However, levels of glutamate pyruvate transaminase, a marker

CMO1 Deficiency Impairs Lipid Metabolism

serum cholesterol ester levels were
specifically increased in CMO1/
mice (Fig. 7, J and K). Consistent
with lipid accumulation in the liver,
the levels of serum FFA were also
significantly increased (Fig. 7L), as
already observed in CMO1/ mice
raised with the fat-unsupplemented
standard chow.

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DISCUSSION
All natural vitamin A derives
from certain carotenoids with provitamin A activity. Although the
role of vitamin A derivatives is well
established in various cell physiological aspects, little is known about
the impact of the provitamin A on
these processes. Recently, two different carotenoid-cleaving enzymes,
CMO1 and CMO2, have been
molecularly identified in mammals,
including humans (2224, 27).
Ubiquitous expression of these
enzymes in tissues suggests that
besides vitamin A bound to RBP4,
circulating carotenoids may tissuespecifically influence vitamin A-dependent processes. We here established a mouse model with a
homozygous genetic disruption of
the CMO1 gene to learn more about
-carotene metabolism and functions. On a diet providing -carotene as major retinoid precursor,
these mutant mice accumulated the
provitamin in large quantities in
several tissues accompanied by a
decrease in vitamin A levels. In controls, no such -carotene accumulation
became detectable, but tissue
FIGURE 7. Serum parameters of wild-type control and CMO1/ mice. A and CF, relevant serum parame- vitamin A levels were significantly
/
ters of energy metabolism in 28-week-old control (gray) and CMO1
(black) mice. Values are means S.D. in
mg/dl (n 8/genotype; four per gender; *, p 0.001). B, glucose clearance assay with 28-week-old CMO1/ higher. This difference between
and control mice. Values are means S.D. (n 4/genotype). G, weight curves of female CMO1/ and control genotypes was already obvious in
mice over a period of 8 weeks (n 5/genotype) subjected to a high fat diet (vitamin A-rich standard chow
supplemented with 30% (w/w) soybean oil). At the beginning of the study, animals were 20 weeks old. Values intestinal -carotene metabolism.
/
mice large
are means of the weight gain S.D. (*, p 0.02) over a period of 8 weeks. H, representative hematoxylin/eosin- Although in CMO1
stained liver sections of 28-week-old mice after high fat feeding; I, total lipid content of the livers (n 5/gen- amounts of dietary -carotene
/
otype; *, p 0.05). JL, serum parameters in 28-week-old female control (gray) and CMO1
(black) mice
subjected to high fat feeding. Values are means S.D. in mg/dl (n 5/genotype; *, p 0.01). Scale bar, 100 M. accumulated, in controls, besides
Chol-Ester, cholesterol esters; GPT, glutamate pyruvate transaminase; TG, triglycerides; wt, wild type.
trace amounts of -carotene, retinoids were found. Consistent with
this lack of intestinal -carotene
types but significantly more in CMO1/ mice (Fig. 7I). As conversion to retinoids, serum levels of the provitamin were
expected, liver histology revealed that hepatocytes contained highly increased in CMO1/ mice and reached levels up to 14
large droplets, which were much more pronounced in number M. This finding clearly indicates that CMO1 in the wild-type
and size in CMO1/ as compared with control animals (n situation already plays a major role in the conversion of -car5/genotype) (Fig. 7H). Analysis of serum lipids revealed that otene to retinoids in the intestine. However, some -carotene
triglycerides were lower than in mice under standard dietary was also absorbed intact in control animals. Thus, widespread
conditions but comparable between genotypes. Interestingly, provitamin A accumulation in CMO1/ mice supports the

CMO1 Deficiency Impairs Lipid Metabolism

33560 JOURNAL OF BIOLOGICAL CHEMISTRY

SCD1 activity plays a role in the onset of diet-induced hepatic

insulin resistance (52), but possible effects of CMO1 deficiency
on the regulation of glucose metabolism require more detailed
analysis in future research.
Several studies demonstrate that RA signaling plays a role for
adipocytes. In cell culture, an enhanced differentiation of adipocytes has been observed in retinol-depleted serum (53). At
the molecular level, a cross-talk between PPAR and RA signaling during adipocyte differentiation is well established (54, 55).
However, not only RA but also retinaldehyde, which directly
derives from -carotene cleavage via CMO1, has been recently
show to antagonize PPAR activity. In vivo, elevated retinaldehyde levels in adipocytes can prevent diet-induced obesity (39).
Indeed, in CMO1 deficiency, mRNA levels of PPAR-regulated
target genes, such as CD36 and FABP4, were significantly
increased in visceral adipose tissues.
Thus far, our data best fit a model in which PPAR-induced
CMO1 expression (i.e. in adipocytes), leads to the production of
-carotene cleavage products, such as retinaldehyde and/or
RA. These -carotene derivatives may fine tune the cross-talk
between nuclear receptors that regulate fatty acid metabolism
(i.e. by antagonizing PPAR activities). In addition, the second
carotenoid cleavage enzyme, CMO2, must be considered in this
process. This enzyme is expressed in the liver and, as shown
here, in adipose tissues. CMO2 catalyzes an eccentric cleavage
of carotenoids. Those carotenoid cleavage products can repress
PPAR and PPAR as well as RXR activities in cell culture (31).
Here, we found no evidence that CMO2 contributes to the conversion of the bulk of -carotene accumulated in CMO1 deficiency. Considering that signaling molecules are required in
very small quantities, we cannot exclude the possibility that
CMO2 activity on -carotene may also influence the activity of
nuclear receptors. Notably, in our study, we used chow diets
containing trace amounts of various naturally occurring carotenoids. Therefore, CMO2 activity on other carotenoids different from -carotene must also be taken into account. Interestingly, it has been recently shown that nonprovitamin A
carotenoids, such as lycopene, can influence PPAR and CMO1
mRNA expression in rats (56).
In summary, our study identifies CMO1 as the key enzyme
for the conversion of -carotene to vitamin A in mammals. In
addition, we provide evidence that carotenoids via carotenoidoxygenases influence lipid metabolism more generally. In the
future, CMO1/ mice represent a valuable model to elucidate
the essential details in this process. Carotenoids in staple food
are the major source for vitamin A in humans, and vitamin A
deficiency is still a global health problem. Therefore, the need
for a better understanding of carotenoid metabolism and functions is of unquestionable relevance for human health.
AcknowledgmentsWe thank Randall Cassada for helpful comments
on the manuscript. We are grateful to Wolfgang Koester (Central Laboratory Unit, University Hospital Freiburg) for determination of
serum lipid levels. We thank the members of the Carotenoid focus
team within the European Nutrigenomic Organization for valuable
discussion.

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idea that -carotene tissues specifically can impact physiological processes. In this context, it is notable that humans naturally
absorb large quantities of -carotene intact and can accumulate
carotenoids in various tissues, including testis, ovary, and
liver, where mRNA of CMO1 was detected (43). Moreover,
the observation that in CMO1 deficiency, -carotene accumulates in large quantities and tissue retinoid levels drop
down rather excludes that the second carotenoid oxygenase,
CMO2, contributes to vitamin A production systemically or
tissue-specifically.
Besides gross impairments in -carotene metabolism,
CMO1 deficiency affected lipid metabolism more generally. In
CMO1/ mice, the total liver lipid content was elevated, and a
fatty liver phenotype was histologically evident with hepatocytes showing large lipid droplets. Liver steatosis in CMO1 deficiency developed independently of the vitamin A status of the
chow. CMO1/ mice showed liver steatosis when liver stores
for vitamin A were depleted under -carotene supplementation but also when raised on a vitamin A-sufficient standard
chow. Under the latter dietary condition, the capacity to store
retinoids was not altered in CMO1/ mice, but liver steatosis
was obvious with increased levels of triglycerides. Thus, we provide evidence that liver steatosis is directly related to CMO1
deficiency. Consistent with this assumption, mice with
impaired liver vitamin A storage show no liver steatosis (35).
On a vitamin A-sufficient chow, liver steatosis became more
pronounced with age. Additionally, we found that CMO1/
mice gained significantly more weight than C57BL/6 controls
when a high fat diet was fed. This weight gain was associated
with a more pronounced fatty liver. C57BL/6 has been shown to
be more susceptible to diet-induced liver steatosis than 129SvJ
mice (44). F2 129SvJxC57BL/6 intercrosses, representing the
genetic background of CMO1/ mice, behave in this respect
like C57BL/6 mice (45). Serum triglyceride levels were comparable between CMO1/ and C57BL/6 control mice both on
the standard and on the fat-supplemented standard chow.
Serum cholesterol ester levels increased in CMO1-deficient
animals fed with the fat-supplemented chow. Most interestingly, serum FFAs were significantly increased in CMO1 deficiency under each dietary condition. Since the rate of hepatic
uptake of FFAs is unregulated, this pathology may cause liver
steatosis in CMO1/ mice.
The CMO1 gene has been identified as a PPAR- and RXRactivated target gene (25). PPAR activates genes involved in
the oxidation of fatty acids in the liver (46), and PPAR activates genes in anabolic lipid pathways, particularly in adipocytes (47). CMO1 activity on -carotene results in retinaldehyde production. This cleavage product can be converted to
RA. In the liver, RA signaling can influence fatty acid metabolism as well as gluconeogenesis (48 50). Our data rather
exclude the possibility that liver steatosis in CMO1/ mice is
caused by major impairments in glucose metabolism.
CMO1/ mice behaved normally in glucose tolerance assays.
Additionally, mRNA levels of ACOX1, essential for fatty acid
oxidation, were up-regulated, and importantly SCD1 mRNA
levels were significantly decreased as compared with controls.
Systemic SCD1 deficiency has been shown to protect against
diet-induced obesity (51). Additionally, in the liver, elevated

CMO1 Deficiency Impairs Lipid Metabolism

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