Human apolipoprotein D overexpression in transgenic mice induces insulin
resistance and alters lipid metabolism
Sonia Do Carmo, David Fournier, Catherine Mounier, and Eric Rassart
Laboratoire de Biologie Mole´culaire, De´partement des Sciences Biologiques, and BioMed, Centre de Recherches
Biome´dicales, Universite´ du Que´bec a` Montre´al, Montreal, Quebec, Canada
Submitted 28 August 2008; accepted in final form 10 January 2009
Do Carmo S, Fournier D, Mounier C, Rassart E. Human apolipopro-
tein D overexpression in transgenic mice induces insulin resistance and alters
lipid metabolism. Am J Physiol Endocrinol Metab 296: E802–E811, 2009.
First published January 27, 2009; doi:10.1152/ajpendo.90725.2008.—
Apolipoprotein D (apoD), a widely expressed lipocalin, has the
capacity to transport small hydrophobic molecules. Although it has
been proposed that apoD may have multiple tissue-specific, physio-
logical ligands and functions, these have yet to be identified. To gain
insight in some of its functions, we generated transgenic mice over-
expressing human apoD (H-apoD) under the control of neuron-
specific promoters. In Thy-1/apoD and NSE/apoD mice, expression of
H-apoD was strong in the nervous system although weakly detected in
peripheral organs such as the liver and blood cells. These mice
displayed not entirely anticipated metabolic defects. Although they
are not obese and have normal lipid concentration in circulation,
Thy-1/apoD and NSE/apoD mice are glucose intolerant, insulin resis-
tant, and develop hepatic steatosis. The steatosis and its associated
insulin resistance are correlated with impairments in hepatic lipogen-
esis. However, they are not strongly related with inflammation. This
impaired insulin response is not caused by a decrease in circulating
leptin or a modulation of adiponectin and resistin levels. These results
suggest that variations in the levels and/or sites of apoD expression
influence the lipid and glucose metabolism, consolidating apoD as a
target for insulin-resistance-related disorders.
hepatic steatosis; inflammation; neuronal promoters
APOLIPOPROTEIN D (apoD) is a member of the lipocalin super-
family of small hydrophobic molecule transporters. apoD was
first detected in 1963, in association with plasma lipoproteins
(4). Since then, apoD was related with many physiological and
pathological conditions and several potential ligands were
identified: arachidonic acid (AA), progesterone, pregnenolone,
bilirubin, cholesterol, and E-3-methyl-2-hexenoic acid (re-
viewed in Ref. 37).
apoD is widely expressed in vertebrates both during devel-
opment and adulthood. In humans, it is poorly expressed in
liver and intestines, the major sites of synthesis of other
apolipoproteins. It is mainly expressed in the adrenal glands,
kidneys, pancreas, placenta, spleen, lungs, ovaries, testes,
brain, peripheral nerves, and cerebrospinal fluid (18). In the
mouse, apoD is mostly expressed in the central nervous system
(CNS; Ref. 43), mainly in glia but also in neurons. It is also
found at high levels in adipose tissue and at lower levels in
other tissues (10, 54). Therefore, it has been proposed that
apoD may have multiple tissue-specific, physiological ligands
and functions (37, 51).
Both the apoD transcript and/or protein are altered in a broad
range of conditions. Those include cellular growth, differenti-
ation, and stress response (16, 17, 27, 36); different types of
cancer (51); several neurological disorders such as Alzheimer’s
disease, stroke (46), schizophrenia (48), and Parkinson’s dis-
ease (32); and animal models of nervous system pathology (8,
47). apoD is also involved in diverse aspects of lipid metabo-
lism. apoD gene polymorphisms affect plasma lipid levels (13)
and can be used as a genetic marker for obesity, hyperinsulin-
emia, and noninsulin-dependent diabetes mellitus (5, 52). Fur-
thermore, apoD may participate in lipid transfer and reverse
cholesterol transport by directly binding cholesterol (34) or by
its association with apoA-I, lecithin-cholesterol acyltrans-
ferase, and cholesteryl ester transfer protein in high-density
lipoprotein (HDL) fractions (44). Because of its capacity to
bind AA, apoD contributes to membrane phospholipid metab-
olism by stabilizing AA levels in cellular membranes (49). An
involvement in lipid redistribution after peripheral nerve injury
was also suggested (8). Moreover, apoD was documented as a
liver X receptor (LXR)-responsive gene (24) and could play an
important role in the modulation of the lipogenesis/lipolysis
balance in adipocytes by transporting ligands for LXR or
peroxisome proliferator-activated receptor (PPAR)-! or partic-
ipating in LXR-dependent reverse cholesterol transport (24).
Finally, apoD may play a role in the control of food intake and
body weight by interacting specifically with the cytoplasmic
portion of the long form of the leptin receptor Ob-Rb (26).
Therefore, it is not surprising to find apoD modulation in
conditions presenting a deregulation of lipid metabolism, such
as Tangier disease (1), familial lecithin-cholesterol acyltrans-
ferase deficiency (2), and mutations in the apoA-I gene (12), or
in conditions leading to lipid accumulation, such as type 2
diabetes (23) and mouse models of Niemann-Pick disease
type C (54).
Abnormal lipid metabolism contributes to several neurode-
generative disorders, including Alzheimer’s and Parkinson’s
diseases. The involvement of apoD in both lipid metabolism
and neurological disorders suggests a central role in neurode-
generation and/or repair. Still, the precise role of apoD remains
undetermined. Recent reports (21, 41, 53) indicate a protective
effect, as it counteracts aging and oxidative stress. In an
attempt to reveal some aspects of the apoD function, we
generated mice overexpressing human apoD (H-apoD) in the
CNS. We chose to overexpress H-apoD because it allows its
discrimination from the endogenous apoD. During the charac-
terization process, we observed that H-apoD overexpression
Address for reprint requests and other correspondence: E. Rassart, De´par-
tement des Sciences Biologiques, Universite´ du Que´bec a` Montre´al, Case
postale 8888, succursale Centre-ville, Montre´al (Que´bec) H3C 3P8 Canada
(e-mail: rassart.eric@uqam.ca).
The costs of publication of this article were defrayed in part by the payment
of page charges. The article must therefore be hereby marked “advertisement”
in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Am J Physiol Endocrinol Metab 296: E802–E811, 2009.
First published January 27, 2009; doi:10.1152/ajpendo.90725.2008.
0193-1849/09 $8.00 Copyright © 2009 the American Physiological Society http://www.ajpendo.orgE802

affects the metabolism of glucose and lipids of these mice in a
promoter’s strength and expression pattern-depending manner
and also expression pattern. These defects correlate with im-
paired hepatic lipogenesis. However, they do not correlate with
increased inflammation or altered adipokine signaling. It there-
fore appears that a modulation of apoD expression in specific
tissues could alter the glucose and lipid metabolisms.
MATERIALS AND METHODS
Animals. All the experimental procedures were approved by the
Animal Care and Use Committee of Universite´ du Que´bec a` Montre´al.
Animals were housed at 24 " 1°C in a 12-h light-dark cycle and fed
a standard rodent chow ad libitum (Charles River rodent chow #5075,
St.-Hubert, QC, Canada) with free access to water. Experiments were
carried out with 11- to 13-mo-old male animals, unless otherwise
noted. Blood samples were collected for hematology and serum
analysis. Tissues were collected, frozen in dry ice, and kept at#80°C.
Sections of tissues were also frozen in HistoPrep frozen tissue em-
bedding medium (Fisher Scientific, Ottawa, ON, Canada) or fixed in
4% paraformaldehyde and embedded in paraffin for histological
analysis.
Generation of H-apoD transgenic mice. The plasmid apoD/bovine
growth hormone (BGH)/pBSSK generated in our laboratory and
comprising the H-apoD coding sequence followed by the BGH
polyadenylation signal in pBluescript II SK (Stratagene-VWR, Ville
Mont-Royal, QC, Canada) was used for the transgene construction.
Briefly, for the Thy-1/apoD construct, the promoter, the first exon, the
first intron, and the 5$-noncoding region of the second exon of the
human Thy-1 gene (%3.5 kb; generous gift from J. Silver, New York
University Medical Center) were cloned upstream of the apoD coding
sequence in plasmid apoD/BGH/pBSSK (21). The neuron-specific
enolase (NSE)/apoD construct was generated similarly with the 5$-
noncoding region of the rat NSE gene (%1.8 kb; a generous gift from
G. Sutcliffe; Scripps Research Institute, La Jolla, CA). Each transgene
fragment was excised from its plasmid by digestion and prepared for
microinjection by agarose gel electrophoresis and extraction with
QIAEX II Gel extraction kit (Qiagen, Mississauga, ON, Canada).
Microinjection into the pronuclei of fertilized C57BL/6 & CBA
zygotes was performed by standard procedures at the McGill Trans-
genic Facility (Montreal, QC, Canada). Transgenic animals were
identified by PCR and Southern blot analysis of genomic DNA
isolated from 3-wk-old mouse tail biopsies. All mice were back-
crossed into C57BL/6 genetic background for at least eight genera-
tions to ensure phenotype stability. Genotyping was performed by
PCR using an apoD-specific primer (5$-CCC AAT CCT CCG GTG
CAG GAG AA-3$) and a BGH-specific primer (5$-GAA GGC ACA
GTC GAG GCT GAT CAG-3$), producing a 0.6-kb fragment in
transgenic mice (Fig. 1C).
Southern blot analysis. BamHI-digested genomic DNA was sepa-
rated on a 0.8% agarose/TAE gel, denatured, transferred to Osmonics
nylon transfer membrane (Fisher Scientific, Ottawa, ON, Canada),
and ultraviolet fixed for 3 min. The membranes were hybridized with
['-32P]dCTP-labeled probe corresponding to each transgene, ex-
posed to Bio-Rad Imaging Screen K and revealed with a Phos-
phorImager (Bio-Rad Molecular Imager FX). Copy standards are
prepared by mixing nontransgenic tail DNA with a known amount
of transgene DNA.
RNA extraction, Northern blot analysis, and semiquantitative RT-
PCR. Extraction of total RNA was performed with the TRIzol reagent
(Invitrogen, Burlington, ON, Canada). Total RNA (10 (g) was
separated on 1.5% (wt/vol) agarose-formaldehyde gels and blotted to
a nylon membrane. The membranes were hybridized with ['-32P]dCTP-
labeled H-apoD, mouse apoE, or mouse GAPDH cDNAs as described
above. Total RNA was also reverse transcribed using Omniscript RT
kit (Qiagen) and amplified with H-apoD, PPAR', PPAR!, fatty acid
synthase (FAS), fatty acid-binding protein (LFABP), sterol regulatory
element-binding protein-1c (SREBP-1c), or hypoxanthine-guanine
phosphoribosyltransferase-specific primers (Table 1). Amplifications
were carried out for 23 cycles for all genes. A control amplification of
reverse transcription reaction without the addition of reverse tran-
scriptase was used to ensure that there was no DNA contamination.
Immunoblotting. Tissues were homogenized in lysis buffer (50 mM
Tris !HCl pH 7.3, 150 mM NaCl, 5 mM EDTA, 0.2% Triton X-100,
and 10% Complete protease inhibitor; Roche, Mississauga, ON,
Canada). After 30 min of incubation at 4°C, lysates were sonicated
and cleared by centrifugation. The protein concentration was deter-
mined using a protein assay reagent (Bio-Rad Laboratories, Missis-
sauga, ON, Canada). All extracts were stored at #80°C. For each
sample, 10 (g of protein were loaded and separated on a 12%
SDS-polyacrylamide gel. The proteins were then transferred to PVDF
membranes blocked with 10% milk and incubated with the primary
antibodies: H-apoD mouse monoclonal antibody (2B9; 1:100,000);
GAPDH rabbit polyclonal antibody (Calbiochem, La Jolla, CA;
1:4,000). These primary antibodies were then detected with the
appropriate horseradish perioxidase-conjugated secondary antibodies
and visualized by chemiluminescence (Amersham ECL, GE-Health-
care, Baie d’Urfe´, QC, Canada) and X-ray film.
Blood analysis. Blood hematology and serum biochemistry were
carried out by the Diagnostic and Research Support Service of McGill
University Animal Resources Center (Montreal, QC, Canada). Levels
of cytokines and adipokines in serum were determined by SearchLight
technology at the SearchLight Sample Testing Service of Pierce
Biotechnology (Woburn, MA). Free fatty acids levels were measured
using a colorimetric kit (MBL International, Woburn, MA).
Competitive ELISA. H-apoD in plasma was quantified by ELISA
using the H-apoD monoclonal antibody 2B9 as previously described
(46). Briefly, microtiter plates were coated with antigen (1 (g apoD/
ml) in 5 mM glycin buffer (pH 9.2) and incubated overnight at 4°C.
Fig. 1. Structure of the human apolipoprotein D (H-apoD) transgene and
analysis of transgenic mice. A: promoter/enhancer region (white box) of the
Thy-1 or neuron-specific enolase (NSE) genes were fused to the H-apoD
coding sequence (black box) followed by the bovine growth hormone (BGH)
polyadenylation signal (gray box). B, BamHI; H, HindIII; K, KpnI; RI, EcoRI;
S/X, SalI/XhoI; X, XbaI. The transgenes were excised by digestion with
EcoRI/XhoI (Thy-1/apoD; 4.5 kbp) or EcoRI/KpnI (NSE/apoD; 3 kbp).
B: Southern analysis of transgenic mice. BamHI-digested genomic DNA (10
(g) was analyzed using the corresponding transgene as a probe. Copy
standards (left) corresponding to 1 (1c) or 10 (10c) copies of the transgenes are
used to estimate the transgene copy number. C: representative PCR genotyping
of the mice. A band (0.6 kbp) is amplified with H-apoD and BGH primers in
the 2 transgenic but not in wild-type (WT) mice.
E803INSULIN RESISTANCE IN APOD TRANSGENIC MICE
AJP-Endocrinol Metab • VOL 296 • APRIL 2009 • www.ajpendo.org