This article is available online at http://www.jlr.org
Journal of Lipid Research Volume 53, 2012
Copyright © 2012 by the American Society for Biochemistry and Molecular Biology, Inc.
Apolipoprotein A-I (apoA-I) mimetic peptides have
been studied in animals and humans for their ability to
improve biomarkers of infl ammation and in animal mod-
els of atherosclerosis for their ability to decrease lesions
( 1 ). The mechanism of action of these peptides has been
thought to be due to their remarkable ability to bind oxi-
dized lipids compared with apoA-I ( 2 ). The classes of oxi-
dized lipids bound with high affi nity by these mimetic
peptides include oxidized phospholipids, oxidized metab-
olites of arachidonic and linoleic acids, and oxidized ste-
rols ( 2 ). Oxidized metabolites of arachidonic and linoleic
acids can be generated by enzymatic systems such as lipox-
ygenases or cyclooxygenases, or they can be generated by
nonspecifi c oxidation. Except for 20-hydroxyeicosatetra-
enoic acid (HETE), all of the oxidized metabolites of
arachidonic and linoleic acids that we studied bound to
the peptide with much higher affi nity than was the case for
the binding to apoA-I ( 2, 3 ).
We recently reported that the dosage of the apoA-I mi-
metic peptide 4F administered to apoE ? / ? mice deter-
mined effi cacy, but plasma and hepatic levels of peptide
did not ( 1 ). Because effi cacy was similar at the same dos-
ages but plasma and hepatic levels were dramatically
higher when the peptide was administered by subcutane-
ous injection (SQ) compared with oral administration, we
suspected that there might be a compartment outside of
the liver or plasma where peptide concentration would be
Abstract To test the hypothesis that intestine is a major
site of action for D-4F, LDLR ? / ? mice were fed a Western
diet (WD) and administered the peptide subcutaneously
(SQ) or orally. Plasma and liver D-4F levels were 298-fold
and 96-fold higher, respectively, after SQ administration,
whereas peptide levels in small intestine only varied by 1.66 ±
0.33-fold. Levels of metabolites of arachidonic and lino-
leic acids known to bind with high affi nity to D-4F were sig-
nifi cantly reduced in intestine, liver and hepatic bile to a
similar degree whether administered SQ or orally. However,
levels of 20-HETE, which is known to bind the peptide with
low affi nity, were unchanged. D-4F treatment reduced
plasma serum amyloid A (SAA) and triglyceride levels ( P <
0.03) and increased HDL-cholesterol levels ( P < 0.04) simi-
larly after SQ or oral administration. Plasma levels of me-
tabolites of arachidonic and linoleic acids signifi cantly
correlated with SAA levels ( P < 0.0001). Feeding 15-HETE
in chow (without WD) signifi cantly increased plasma SAA
and triglyceride levels and decreased HDL-cholesterol and
paraoxonase activity ( P < 0.05), all of which were signifi -
cantly ameliorated by SQ D-4F ( P < 0.05). We conclude
that D-4F administration reduces levels of free metabolites
of arachidonic and linoleic acids in the small intestine
and this is associated with decreased infl ammation in LDL
receptor defi cient mice . —Navab, M., S. T. Reddy, G. M.
Anantharamaiah, G. Hough, G. M. Buga, J. Danciger, and A.
M. Fogelman. D-4F-mediated reduction in metabolites of
arachidonic and linoleic acids in the small intestine is associ-
ated with decreased infl ammation in low-density lipoprotein
receptor-null mice. J. Lipid Res . 2012. 53:437–445.
Supplementary key words apolipoprotein A-I • apolipoprotein A-I mi-
metic peptides • lipoproteins • hydroxyeicosatetraenoic acid • hy-
droxyoctadecadienoic acid • small intestine
This work was supported in part by US Public Health Service Grants HL-30568,
HL-34343, and the Laubisch, Castera, M.K. Grey Funds at UCLA and the
Leducq Foundation. MN, STR, GMA, and AMF are principals in Bruin
Pharma. AMF is an offi cer in Bruin Pharma.
Manuscript received 7 August 2011 and in revised form 11 December 2011.
Published, JLR Papers in Press, December 13, 2011
D-4F-mediated reduction in metabolites of arachidonic
and linoleic acids in the small intestine is associated
with decreased infl ammation in low-density lipoprotein
Mohamad Navab , * ,1 Srinivasa T. Reddy , * G.M. Anantharamaiah , † Greg Hough , *
Georgette M. Buga , * Jan Danciger , * and Alan M. Fogelman *
*Department of Medicine, David Geffen School of Medicine at UCLA , Los Angeles, CA ;
† Department of Medicine, University of Alabama at Birmingham , AL
Abbreviations: apoE ? / ? , ApoE null mice; D-4F, the peptide Ac-D-
W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH 2 synthesized from all D-amino
acids; HETE, hydroxyeicosatetraenoic acid; HODE, hydroxyoctadeca-
dienoic acid; LDLR ? / ? , low-density lipoprotein receptor null mice; PG,
prostaglandin; SAA, serum amyloid A; SQ, subcutaneous injection; TX,
thromboxane; WD, Western diet.
1 To whom correspondence should be addressed.
The online version of this article (available at http://www.jlr.org)
contains supplementary data in the form of three tables and one
by guest, on October 27, 2015
Supplemental Material can be found at:
438Journal of Lipid Research Volume 53, 2012
from Cayman Chemical Co. (Ann Arbor, MI). All other materials
were purchased from sources previously described ( 2, 3, 7 ).
Female LDLR ? / ? mice originally purchased from Jackson lab-
oratories on a C57BL/6J background were obtained from the
breeding colony of the Department of Laboratory and Animal
Medicine at the David Geffen School of Medicine at UCLA. The
mice used in these studies were of different ages, which are stated
in each Table legend. The mice were maintained on a chow diet
(Ralston Purina) before being switched to a WD (Teklad, Har-
lan, catalog # TD88137). The WD was kept frozen until it was
administered to the mice each night. Peptide was administered
to the mice by daily SQ) on the back or was administered orally
by providing the peptide in the drinking water. For oral adminis-
tration, the peptide was freshly prepared by dissolving in water,
and the drinking water was changed three times per week. For
SQ administration, the peptide was uniformly dissolved in nor-
mal saline (pH 7.4) using a glass-glass homogenizer, and each
mouse received a daily injection of 1 ml containing the peptide
in normal saline (pH 7.4). In all experiments, the daily dose
of peptide administered by either route of administration was
900 ? g/mouse/day (45 mg/kg/day).
Addition of oxidized fatty acids to mouse chow
The following protocol was used to conveniently and accu-
rately provide free oxidized fatty acids in the chow diet. Three
hundred milliliters of water were added to 400 g of chow pellets.
The pellets were allowed to soften for 30 min, and a highly uni-
form mixture was generated using a small industrial-type mixer.
The desired quantity of the free oxidized fatty acid was fi rst mixed
in saline and was then carefully and gradually added and mixed
into the chow using the mixer. Mixing was continued at high
speed for 1 min, at which time the mixer was stopped and the
material was mixed in the opposite direction using a spatula to
ensure a highly uniform distribution of the added free oxidized
fatty acid. This process was continued for a total of 5–10 cycles.
The resulting mixture containing the oxidized fatty acid was
spread and fl attened uniformly on a sheet of aluminum foil in a
tray to achieve a height of ? 2 mm. The mixture was then cut into
blocks of ? 5 g each using a rotary knife. These blocks were stored
at ? 20°C. Each evening, four blocks of the frozen diet contain-
ing the oxidized fatty acids were provided to each cage contain-
ing four mice. The mice ate all of the diet by morning. The frozen
diet contained 1 ? g free oxidized fatty acid per gram frozen diet.
Thus, on average each mouse received 5 ? g of the free oxidized
fatty acid each day.
All experiments were performed using protocols approved by
the Animal Research Committee at UCLA.
Determination of free arachidonic acid and free
metabolites of arachidonic and linoleic acids in plasma,
liver, hepatic bile, and small intestine and in the contents
of the small intestine
The levels of free arachidonic acid and free metabolites of
arachidonic and linoleic acids were determined by LC-ESI-MS/MS
as previously described ( 3 ). In each instance, a deuterium-
labeled internal standard was included to correct for extraction
effi ciency and to facilitate quantifi cation ( 3 ).
Preparation and analysis of plasma. After an overnight fast,
mouse plasma was immediately prepared from heparinized
blood obtained from the retroorbital sinus under mild isofl u-
rane anesthesia. For mice receiving subcutaneous injections,
the blood was removed 1.5–3 h after the last injection. Plasma
similar. We found that the concentration of D-4F in the
feces was the same regardless of whether the peptide was
administered SQ or orally, suggesting that the intestine
maybe a major site of action for the peptide regardless of
the route of administration ( 1 ).
The concentration of free 15-HETE and 13-hydroxyoc-
tadecadienoic acid (HODE) in the plasma of apoE ? / ?
mice was signifi cantly higher than that of wild-type mice
( 3 ). After administration of the 4F peptide, plasma levels
of free oxidized fatty acids that bound with higher affi nity
to the mimetic peptide compared with apoA-I (e.g.,
5-HETE, 15-HETE, 9-HODE, and 13-HODE) signifi cantly
decreased, but the levels of 20-HETE, which bound with
equal low affi nity to apoA-I and 4F, did not decrease ( 3 ).
These studies focused on the plasma levels of free oxidized
fatty acids, which are only a small fraction (<10%) of the
total plasma oxidized fatty acids. Interestingly, only the
free oxidized fatty acid plasma levels decreased after the
administration of the apoA-I mimetic peptide; esterifi ed
oxidized fatty acid levels were unchanged ( 3 ).
In other studies, apoE ? / ? mice were made diabetic, re-
sulting in a signifi cant increase in the hepatic content of
free arachidonic acid and free 12-HETE, 15-HETE, 13-
HODE, PGD 2 , and PGE 2 . This was associated with a signifi -
cant increase in aortic atherosclerosis. Oral administration
of D-4F signifi cantly decreased the hepatic content of free
arachidonic acid and free oxidized fatty acids derived from
arachidonic and linoleic acids and signifi cantly decreased
aortic atherosclerosis without affecting other plasma lipid
or lipoprotein levels ( 4 ).
Subsequently, we reported that HDL from type 2 dia-
betics contained signifi cantly more free 5-HETE, 12-
HETE, 15-HETE, 9-HODE, and 13-HODE than HDL
from healthy volunteers. The type 2 diabetic HDL was
also proinfl ammatory in a cell-based assay and was abnor-
mal in a cell-free assay. The HDL content of free 5-HETE,
12-HETE, 15-HETE, 9-HODE, and 13-HODE signifi cantly
correlated with the values obtained in the cell-free assay
( 5 ).
The experiments reported here were designed to ex-
tend our previous studies on the interaction of the D-4F
peptide with the intestine ( 1 ). These studies confi rm our
previous studies ( 1–5 ) and demonstrate for the fi rst
time that D-4F-mediated reductions in free oxidized
metabolites of arachidonic and linoleic acids in the small
intestine are associated with reduced infl ammation in
LDLR ? / ? mice.
MATERIALS AND METHODS
The peptide 4F (Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH 2 )
was synthesized from all D-amino acids (D-4F) by solid-phase syn-
thesis as described ( 6 ). Protease inhibitors were purchased from
Roche Diagnostics Gmbh (Complete, Mini, EDTA-free tablet)
(Catalogue No. 11 836 179 001). In the experiments in which a
HETE or HODE were added to mouse chow without the Western
diet (WD), 12( S )-HETE (Catalog #34570), 15( S )-HETE (Catalog
#34720), and 13( S )-HODE (Catalog #38610) were purchased
by guest, on October 27, 2015
Supplemental Material can be found at:
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by guest, on October 27, 2015
Supplemental Material can be found at: