Increased CYP2J3 expression reduces insulin resistance in fructose-treated rats and db/db mice.

Abstract

Accumulating evidence suggests that cytochrome P450 (CYP) epoxygenases metabolize arachidonic acid into epoxyeicosatrienoic acids (EETs), which play crucial and diverse roles in cardiovascular homeostasis. The anti-inflammatory, antihypertensive, and pro-proliferative effects of EETs suggest a possible beneficial role for EETs on insulin resistance and diabetes.
This study investigated the effects of CYP2J3 epoxygenase gene therapy on insulin resistance and blood pressure in diabetic db/db mice and in a model of fructose-induced hypertension and insulin resistance in rats.
CYP2J3 gene delivery in vivo increased EET generation, reduced blood pressure, and reversed insulin resistance as determined by plasma glucose levels, homeostasis model assessment insulin resistance index, and glucose tolerance test. Furthermore, CYP2J3 treatment prevented fructose-induced decreases in insulin receptor signaling and phosphorylation of AMP-activated protein kinases (AMPKs) in liver, muscle, heart, kidney, and aorta. Thus, overexpression of CYP2J3 protected against diabetes and insulin resistance in peripheral tissues through activation of insulin receptor and AMPK pathways.
These results highlight the beneficial roles of the CYP epoxygenase-EET system in diabetes and insulin resistance.

Full text

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Increased CYP2J3 Expression Reduces Insulin
Resistance in Fructose-Treated Rats and db/db Mice
Xizhen Xu,1Chun Xia Zhao,1Luyun Wang,1Ling Tu,1Xiaosai Fang,1Changlong Zheng,1
Matthew L. Edin,2Darryl C. Zeldin,2and Dao Wen Wang1
OBJECTIVE—Accumulating
chrome P450 (CYP) epoxygenases metabolize arachidonic acid
into epoxyeicosatrienoic acids (EETs), which play crucial and
diverse roles in cardiovascular homeostasis. The anti-inflamma-
tory, antihypertensive, and pro-proliferative effects of EETs
suggest a possible beneficial role for EETs on insulin resistance
and diabetes.
evidencesuggests thatcyto-
RESEARCH DESIGN AND METHODS—This study investi-
gated the effects of CYP2J3 epoxygenase gene therapy on insulin
resistance and blood pressure in diabetic db/db mice and in a
model of fructose-induced hypertension and insulin resistance in
rats.
RESULTS—CYP2J3 gene delivery in vivo increased EET gener-
ation, reduced blood pressure, and reversed insulin resistance as
determined by plasma glucose levels, homeostasis model assess-
ment insulin resistance index, and glucose tolerance test. Further-
more, CYP2J3 treatment prevented fructose-induced decreases
in insulin receptor signaling and phosphorylation of AMP-acti-
vated protein kinases (AMPKs) in liver, muscle, heart, kidney,
and aorta. Thus, overexpression of CYP2J3 protected against
diabetes and insulin resistance in peripheral tissues through
activation of insulin receptor and AMPK pathways.
CONCLUSIONS—These results highlight the beneficial roles of
the CYP epoxygenase-EET system in diabetes and insulin
resistance. Diabetes 59:997–1005, 2010
A
cyclooxygenase, the lipoxygenase, and the cytochrome
P450 (CYP) epoxygenase pathways. Metabolism of arachi-
donic acid via CYP epoxygenases produces four different
cis-epoxyeicosatrienoic acids (EETs): 5,6-, 8,9-, 11,12-, and
14,15-EET. Human P450 2J2 (CYP2J2) and its rat homolog
CYP2J3 are predominant enzymes responsible for the
oxidation of endogenous arachidonic acid pools in cardiac
myocytes, vascular endothelium, pancreas, and other tis-
rachidonic acid is liberated from cell mem-
branes by phospholipases in response to vari-
ous stimuli and is subsequently metabolized by
three different enzyme pathways, namely the
sues where they exert regulatory effects in normal and
pathophysiological processes (1–4).
Accumulating evidence suggests that EETs play crucial
and diverse roles in cardiovascular homeostasis. Arachi-
donic acid epoxygenase metabolites stimulate endothelial
cell growth and angiogenesis via mitogen-activated protein
kinase (MAPK) and phosphatidylinositol 3-kinase (PI 3-ki-
nase)/AKT signaling pathways, and to some extent, the
endothelial NO synthase (eNOS) pathway (5). Moreover,
EETs upregulate eNOS in bovine aortic endothelial cells
via activation of MAPK, protein kinase C, PI 3-kinase/AKT,
and MAPK signaling pathways (6,7). CYP epoxygenase
overexpression, which increases EET biosynthesis, signif-
icantly protects endothelial cells from apoptosis induced
by tumor necrosis factor-? (TNF-?), an effect that is
mediated, at least in part, through inhibition of MAPK
dephosphorylation and activation of PI 3-kinase/AKT path-
ways (8). Furthermore, CYP-derived eicosanoids are vaso-
dilatory, at least in part through their ability to activate
eNOS and NO release (9).
Considerableexperimental
eNOS-derived NO is a pivotal regulator of blood pressure,
vascular tone, and vascular homeostasis (10–14). Experi-
mental evidence also suggests that NO is involved in the
pathogenesis of diabetes and insulin resistance. NADPH
oxidases in the vascular wall are activated in diabetes,
leading to enhanced degradation of NO and the production
of reactive oxygen species (15). Uncoupling of eNOS has
been demonstrated in animal models of diabetes (16), and
endogenous NO synthase inhibitors including asymmetric
dimethylarginine are an important cause of vascular insu-
lin resistance (17). Cumulatively, these data indicate that
diabetes and insulin resistance are characterized, to some
degree, by endothelial dysfunction, altered eNOS expres-
sion, and NO production. Insulin mediates its effects
through binding to insulin receptors and triggering down-
stream signaling pathways, of which the most important is
the PI 3-kinase/AKT pathway. This pathway is involved in
a variety of insulin responses including protection from
apoptosis and transport of glucose through cell mem-
branes in endothelial cells (8,18).
We hypothesized that overexpression of CYP2J3 and
the subsequent increase in production of EETs might be
beneficial in attenuating hypertension and insulin resis-
tance. Thus, the present study investigated the effects and
underlying mechanisms of CYP2J3 gene therapy on insulin
resistance and diabetes in fructose-induced insulin resis-
tance in rats and in diabetic db/db mice.
evidencesuggeststhat
RESEARCH DESIGN AND METHODS
Materials and reagents. Materials were obtained from the following suppli-
ers: Antibodies involved in this study were from Santa Cruz Biotechnology
(Santa Cruz, CA); the rabbit polyclonal anti-CYP2J3 antibody was developed
in our laboratory as described (4); fructose, glucose, triglyceride, and choles-
From the1Department of Internal Medicine and The Institute of Hypertension,
Tongji Hospital, Tongji Medical College of Huazhong University of Science
and Technology, Wuhan, People’s Republic of China; and the2Division of
Intramural Research, National Institute of Environmental Health Sciences,
National Institutes of Health, Research Triangle Park, North Carolina.
Corresponding author: Dao Wen Wang, dwwang@tjh.tjmu.edu.cn.
Received 27 August 2009 and accepted 21 December 2009. Published ahead of
print at http://diabetes.diabetesjournals.org on 12 January 2010. DOI:
10.2337/db09-1241.
X.X., C.X.Z., and L.W. contributed equally to this study.
© 2010 by the American Diabetes Association. Readers may use this article as
long as the work is properly cited, the use is educational and not for profit,
and the work is not altered. See http://creativecommons.org/licenses/by
-nc-nd/3.0/ for details.
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.
ORIGINAL ARTICLE
diabetes.diabetesjournals.orgDIABETES, VOL. 59, APRIL 2010997

Page 2

terol reagents were from Ningbo Cicheng Biocompany (Ningbo, China);
Rat/Mouse Insulin ELISA Kit was from Linco Research (St. Charles, MO);
14,15-DHET ELISA Kit was from Detroit R&D (Detroit, MI); and guanosine
3?,5?-cyclic monophosphate (cGMP) and cAMP ELISA kit was from Cayman
Chemical (Ann Arbor, MI). All other chemicals and reagents were purchased
from Sigma-Aldrich unless otherwise specified. Full-length CYP2J3 cDNA was
cloned from rat liver RNA and then subcloned into the pcDNA plasmid vector
in sense (CYP2J3?) and antisense (CYP2J3?) orientations.
Animals. All animal experimental protocols complied with standards stated
in the National Institutes of Health Guidelines for the Care and Use of
Laboratory Animals and were approved by The Academy of Sciences of
China. Male Sprague-Dawley rats weighing 200 ? 20 g and male C57BL/6 and
db/db mice (8 weeks old) were obtained from the Experimental Animal Center
of Shanghai (Shanghai, People’s Republic of China). Animals were treated
before experiments as described previously (19).
Fructose feeding and gene delivery protocols. After a 1-week adaptation
period (i.e., beginning at week 0), rats were fed normal rat chow and either
normal water (n ? 32) or water containing 10% fructose (n ? 24) for a total of 5
weeks. Systolic blood pressure was measured weekly until week 6, and gene
delivery protocols were undertaken at week 3 as described previously (19).
For the mouse study, C57BL/6 and db/db mice were anesthetized with
diethyl ether and received a sublingual vein injection of plasmid at a dose of
5 mg/kg body wt. C57BL/6 mice received either empty pcDNA (C57BL/6 ?
pcDNA), CYP2J3?(C57BL/6 ? CYP2J3?), or 0.9% NaCl (C57BL/6 normal)
(n ? 8 per group), and db/db mice similarly received either pcDNA (db/db ?
pcDNA), CYP2J3?(db/db ? CYP2J3?), or 0.9% NaCl (db/db normal) (n ? 8 per
group).
Blood pressure measurement. Systolic blood pressure was measured
weekly in conscious rats with a manometer-tachometer (Rat Tail NIBP
System; ADI Instruments, Bella Vista, NSW, Australia) using the tail-cuff
method as described previously (19).
Rat serum and urine analyses. Serum and urine samples from all rats were
collected. Fasting serum levels of insulin, glucose, sodium, potassium, mag-
nesium, cholesterol, triglycerides, LDL cholesterol, and HDL cholesterol as
well as urine levels of sodium, potassium, and magnesium were assessed as
described previously (19). Similarly, serum and urine samples from all mice
were collected, and serum levels of insulin, glucose, cholesterol, triglycerides,
LDL cholesterol, and HDL cholesterol were assessed. Serum levels of alanine
aminotransferase, urea, and creatinine in rats were also measured on an
AEROSET Clinical Chemistry System (Abbott Laboratories) to evaluate renal
and liver function. Additional details are provided in the supplementary
methods, available in an online appendix at http://diabetes.diabetesjournals.
org/cgi/content/full/db09-1241/DC1.
Glucose tolerance test. At 2 weeks after gene delivery, C57BL/6 and db/db
mice were fasted overnight (for 16 h) and then injected intraperitoneally with
D-glucose (20% solution; 2 g/kg body wt). At 0, 30, 60, and 120 min after glucose
administration, blood samples were taken from the cavernous sinus with a
capillary while under ether anesthesia. Plasma glucose and insulin levels were
determined using methods identical to those used for serum samples in rats
(described in detail in the online supplementary methods).
Evaluation of urine 14,15-DHET by ELISA. To assess in vivo EET
production, an ELISA kit (Detroit R&D) was used to determine concentrations
of the stable EET metabolite 14,15-dihydroxyeicosatrienoic acid (14,15-
DHET) in the urine of rats and mice. 14,15-DHET was quantified by enzyme-
linked immunosorbent assay (ELISA) according to the manufacturer’s
instructions, as previously described (7).
RT-PCR analysis of aortic ET-1 and ETA-R mRNA. Total RNA was
extracted from frozen rat aortas using TRIZOL reagent (Invitrogen, Carlsbad,
CA) and used to assess mRNA levels of ET-1 and ETA-R. The following
oligonucleotide primers were used for amplification of the ET-1 and ETA-R
cDNAs from reverse-transcribed aortic RNA: ET-1 (forward): 5?-AAGCGTT-
GCTCCTGCTCCTCC-3?; ET-1 (reverse): 5?-TTCCCTTGGTCTGGTCTTTGTG-
3?; ETA-R (forward): 5?-TGCTCAACGCCACGACCAAGT-3?; ETA-R (reverse):
5?-GGTGTTCGCTGAGGGCAATCC-3?.
Amplification was performed on an ABI7500 PCR system (Applied Biosys-
tems, Darmstadt, Germany) after incubation with Moloney murine leukemia
virus reverse transcriptase at 42°C for 15 min. A preheating step of 5 min at
95°C was followed by 40 cycles consisting of 30 s at 95°C, 20 s at 60°C, and 20 s
at 72°C. PCR products were electrophoresed on 1.5% agarose gels. The
quantities of specific ET-1 and ETA-R transcripts were normalized to levels of
glyceraldehyde-3-phosphate dehydrogenase transcripts to control for RNA
quality and amount.
Western blot analysis. At 2 weeks after gene injection, rats and mice from
each group were anesthetized with pentobarbital (100 mg/kg i.p.), and skeletal
muscles,aortas,hearts,kidneys,andliverswereexcised,frozeninliquidnitrogen,
and stored at ?80°C. Western blotting was performed as described previously
(19). Expression was quantified by densitometry and normalized to ?-actin
expression. All groups were then normalized to their respective controls, and bar
graphs represent quantification of at least three independent experiments.
Evaluation of urinary cAMP and cGMP by ELISA. Urinary cAMP and
cGMP levels were measured by ELISA as previously described (20,21).
Statistical analysis. Continuous data were expressed as means ? SEM.
Comparisons between groups were performed by a one-way ANOVA. Two-
way ANOVA was used to examine differences in response to treatments and
between groups, with post hoc analyses performed using the Student-
Newman-Keuls test. Statistical significance was defined as P ? 0.05.
RESULTS
Blood pressure and metabolic changes in fructose-
treated rats. All rats in the study were assessed for a
variety of physiological parameters 3 weeks after receiving
either control or fructose-containing drinking water. As
expected, consumption of fructose-containing water re-
sulted in significantly increased systolic blood pressure,
significantly increased levels of serum insulin, serum tri-
glyceride, urine potassium, urine magnesium, and urine
volume, and significantly decreased urine osmolarity (all
P ? 0.05) (Table 1, Fig. 1, and supplementary Table 1).
Homeostasis model assessment insulin resistance (HOMA-
IR) also was significantly increased in fructose-treated rats
(P ? 0.05) (Table 1). These data indicate that fructose
administration induced hypertension, insulin resistance,
and hypo-osmolar diuresis as described previously (19).
Effects of CYP2J3 gene delivery on CYP2J3 protein
expression and on fructose-induced hypertension
and pathophysiological changes in rats. At 2 weeks
after gene delivery (i.e., at week 5 of the study), CYP2J3
protein levels were increased in the aorta, heart, liver, and
kidney of fructose-treated CYP2J3?(F ? CYP2J3?) rats
compared with fructose-treated rats injected with CYP2J3?
or with the empty pcDNA vector (Fig. 1A). Importantly,
CYP2J3 functionality was demonstrated by the nearly four-
fold increase in urinary 14,15-DHET levels in rats injected
with CYP2J3?compared with those injected with CYP2J3?
or with the empty pcDNA vector (Fig. 1B).
Injection of CYP2J3?to fructose-treated rats resulted in
decreased systolic blood pressure 1, 2, and 3 weeks after
injection (i.e., at weeks 4, 5, and 6 of the study, respec-
tively) compared with that observed in fructose-treated
rats injected with the control pcDNA or CYP2J3?vectors
(Fig. 1C). The maximum reduction in blood pressure in F
? CYP2J3?rats was observed 2 weeks after injection (i.e.,
at week 5 of the study) when blood pressure reached a
level similar to those observed in rats drinking normal
water (Fig. 1C). No changes in blood pressure compared
TABLE 1
Physiological parameters determined in rats after 3 weeks of
administration of control or fructose-containing drinking water
Variables
Control group
(n ? 32)
Fructose-treated
group (n ? 24)
Glucose (mmol/l)
Triglycerides (mmol/l)
Insulin (mIU/l)
HOMA-IR
Body weight (g)
4.91 ? 0.2
0.45 ? 0.12
17.74 ? 0.59
4.21 ? 0.56
254 ? 33
5.62 ? 0.4
1.18 ? 0.47*
49.11 ? 0.42†
14.24 ? 0.86†
267 ? 26
Physiological parameters determined in rats after 3 weeks of admin-
istration of normal or fructose-containing drinking water. Fasting
triglyceride, insulin levels, and HOMA-IR were increased in fructose-
treated rats after 3 weeks of administration of fructose-containing
drinking water. Values shown are mean ? SE from each group of
rats, respectively. *P ? 0.05, †P ? 0.001 compared with rats treated
with water.
CYP2J3 ATTENUATES INSULIN RESISTANCE
998DIABETES, VOL. 59, APRIL 2010 diabetes.diabetesjournals.org

Page 3

with saline-injected rats were observed in normal drinking
water–treated rats administered pcDNA, CYP2J3?, or
CYP2J3?(Fig. 1C). These data indicate that CYP2J3 overex-
pression reduced hypertension in fructose-treated rats but
had no effect on blood pressure in normal control rats.
Other physiological and biochemical parameters related
to hypertension and hyperinsulinemia were assessed in
eight rats per experimental group 2 weeks after gene
delivery (week 5) (Table 2 and supplementary Table 2).
Compared with values in the normal water–treated
groups, serum insulin, insulin resistance (HOMA-IR), se-
rum triglycerides, urine volume, urine potassium, and
urine magnesium levels were all higher, whereas urine
osmolarity was lower in fructose-treated rats injected with
the empty pcDNA vector (F ? pcDNA group; all P ? 0.05)
(Table 2 and supplementary Table 2). With the exception
of serum triglyceride levels, all of these changes were
prevented by injection of fructose-treated rats with
CYP2J3?, but not with CYP2J3?(Table 2 and supplemen-
tary Table 2). These data indicate that CYP2J3 overexpres-
sion markedlyattenuated
resistance in rats but that it had no effect on these
parameters in normal water–treated rats.
Compared with values in the normal water–treated
groups, serum alanine aminotransferase, urea nitrogen, and
creatinine showed no significant changes in fructose-treated
rats injected with the empty pcDNA vector (F ? pcDNA
group; all P ? 0.05). Compared with values in rats injected
with empty pcDNA vector, serum alanine aminotransferase,
urea nitrogen, and creatinine were not significantly changed
in rats injected with CYP2J3?(supplementary Table 3).
These data indicate that CYP2J3 overexpression has no
detrimental effects on rat renal and liver function.
Effects of CYP2J3 gene delivery on rat insulin recep-
tor signaling and eNOS, ET-1, and ETA-R expression.
PI 3-kinase is recruited to insulin receptor substrate (IRS)
signaling complexes through binding of Src homology 2
domains in its 85-kDa regulatory subunit to specific phos-
photyrosine residues in IRS-1, a process that leads to
activation of the PI 3-kinase p110 catalytic subunit (22). To
investigate the signaling mechanisms through which
CYP2J3 attenuates fructose-induced insulin resistance, we
evaluated the expression of IRS-1 associated with the
insulin signaling cascade in rat liver and skeletal muscle.
Compared with levels in normal control rats, fructose
drinking resulted in significantly decreased IRS-1 levels in
liver and skeletal muscle (Fig. 2A and B). Phospho-Y989-
IRS-1 levels were similarly decreased in liver and
skeletal muscle, whereas phospho-S307-IRS-1 was sig-
nificantly increased (supplementary Fig. 1A and B).
Administration of CYP2J3?significantly reversed the
changes in IRS-1 and phospho–IRS-1 levels induced by
fructose in both liver and skeletal muscle (Fig. 2A and B;
supplementary Fig. 1A and B).
Compared with expression in corresponding control
animals, eNOS protein expression was downregulated in
aorta, liver, and skeletal muscle in fructose-treated rats;
this effect was not observed in fructose-treated rats in-
jected with CYP2J3?(Fig. 2C; supplementary Fig. 1C and
D). Similar changes in eNOS expression and effects of
CYP2J3?were observed in rat heart and kidney (data not
shown). These data suggest that CYP2J3?treatment re-
stored eNOS activity and support previous observations of
a beneficial effect of eNOS against fructose-induced hyper-
tension and hyperinsulinemia (23).
The expression of ET-1 and ETA-R mRNA transcripts in
fructose-inducedinsulin
A
C
B
CYP2J3
β-actin
pcDNA
CYP2J3(-)
CYP2J3(+)
* **
AK H LAKLHAK H L
Fructose
pcDNA
CYP2J3(+)
CYP2J3(-)
14,15-DHET (ng/ml)
0
20
40
60
-
+
-
-
-
-
+
-
-
-
-
+
-
-
-
-
-
-
-
-
+
-
+
-
+
+
+
+
*
*
#
#
N + pcDNA
N + CYP2J3(+)
N + CYP2J3(-)
Normal
F + pcDNA
F + CYP2J3(+)
F + CYP2J3(-)
Systolic BP (mmHg)
110
120
115
130
125
140
135
105
1
2
3
40
5
6
Weeks
*
#
*
**
*
#
**
*
#
**
*
*
0
1
2
3
4
5
A
H
L
K
*
*** *
#
#
# #
pcDNA
CYP2J3(-)CYP2J3(+)
CYP2J3/β-actin
(Relative Intensity)
FIG. 1. Effects of CYP2J3 gene delivery on CYP2J3 protein expression,
urinary 14,15-DHET levels, and fructose-induced hypertension in rats.
A: CYP2J3 protein levels were increased in aorta (A), heart (H),
liver (L), and kidney (K) of fructose-treated rats 2 weeks after
injection of CYP2J3?but not after injection of CYP2J3?or empty
vector (pcDNA). B: Urinary 14,15-DHET levels were increased in
control and fructose-treated rats injected with CYP2J3?compared
with rats injected with CYP2J3?or empty vector (pcDNA). *P < 0.01
versus pcDNA; #P < 0.01 versus CYP2J3?; n ? 8 per group. C: The
elevation of systolic blood pressure (BP) observed in fructose-
treated rats was decreased 1, 2, and 3 weeks after injection of
CYP2J3?(i.e., at weeks 4, 5, and 6 of the study, respectively) but not
after injection with CYP2J3?or empty vector (pcDNA). Values
shown are mean ? SEM from each group of rats. *P < 0.01 compared
with rats treated without fructose, #P < 0.01 compared with rats
treated with fructose, **P < 0.01 compared with rats treated with
CYP2J3?; n ? 4–10 per group per time point.
X. XU AND ASSOCIATES
diabetes.diabetesjournals.org DIABETES, VOL. 59, APRIL 2010 999

Page 4

rat aortas was determined by RT-PCR 2 weeks after gene
delivery to examine the effects of fructose feeding and
CYP2J3 gene delivery on the endothelin pathway, which
has been shown to play a role in blood pressure homeosta-
sis. Fructose administration resulted in significant in-
creases in aortic ET-1 and ETA-R mRNA levels (Fig. 2D
and E). These changes were prevented in rats adminis-
tered CYP2J3?, but not in rats administered CYP2J3?.
Effects of CYP2J3 gene delivery on CYP2J3 protein
expression and on pathophysiological changes and
glucose tolerance in db/db mice. Similar to observa-
tions in fructose-fed rats, levels of CYP2J3 protein were
increased in the aorta, heart, liver, and kidney in diabetic
db/db mice 2 weeks after injection with CYP2J3?com-
pared with levels observed after injection with the empty
pcDNA vector (Fig. 3A). Functionality of the CYP2J3
TABLE 2
Physiological parameters determined in rats 2 weeks after injection of empty pcDNA3.1 vector, pcDNA-2J3?, or pcDNA-2J3?
Variables
Treatment group
Normal water treated
N ? p2J3?
Normal
Fructose treated
F ? p2J3?
N ? pcDNA3.1
N ? p2J3?
F ? pcDNA3.1N ? p2J3?
Glucose (mmol/l)
Triglycerides (mmol/l)
Insulin (mIU/l)
HOMA-IR
Body weight (g)
5.36 ? 0.68
0.61 ? 0.22
22.65 ? 0.94
5.26 ? 0.82
310 ? 34
6.99 ? 0.06
0.63 ? 0.39
18.16 ? 0.81
4.16 ? 0.36
311 ? 49
5.69 ? 0.86
0.45 ? 0.18
18.83 ? 0.44
6.64 ? 0.61
307 ? 35
5.09 ? 0.36
0.53 ? 0.09
20.5 ? 0.64
5.06 ? 0.26
299 ? 40
6.55 ? 0.58
1.04 ? 0.89*
49.71 ? 0.71*
14.25 ? 0.02*
297 ? 49
6.44 ? 0.68
1.30 ? 0.13
19.48 ? 0.18†
5.40 ? 0.59†
303 ? 31
6.85 ? 0.99
1.26 ? 0.4
60.25 ? 0.08‡
24.28 ? 0.04‡
341 ? 65
Compared with values in the normal water–treated groups, serum insulin, insulin resistance (HOMA-IR), and serum triglycerides levels were
all higher in fructose-treated rats injected with the empty pcDNA vector (F ? pcDNA group). With the exception of serum triglyceride levels,
all of these changes were prevented by injection of fructose-treated rats with CYP2J3?but not CYP2J3?. *P ? 0.05 vs. N ? pcDNA3.1 group;
†P? 0.05 vs. F ? pcDNA3.1 group; ‡P ? 0.05 vs. F ? p2J3?group; n ? 8 per group.
C
eNOS
β-actin
Fructose
pcDNA
CYP2J3(+)
CYP2J3(-)
1.25
1.00
0.75
0.50
0.25
-
+
-
-
-
-
+
-
-
-
-
+
-
-
-
-
-
-
-
-
+
-
+
-
+
+
+
+
0
#
IRS-1
β-actin
IRS-1
β-actin
Fructose
pcDNA
CYP2J3(+)
CYP2J3(-)
Liver IRS1/-β-actin
(Relative Intensity)
Muscle IRS1/-β-actin
Fructose
pcDNA
CYP2J3(+)
CYP2J3(-)
E
(Relative Intensity)
Aorta eNOS/-β-actin
(Relative Intensity)
1.25
1.00
0.75
0.50
0.25
-
+
-
-
-
-
+
-
-
-
-
+
-
-
-
-
-
-
-
-
+
-
+
-
+
+
+
+
0
**
*
#
1.25
1.00
0.75
0.50
0.25
-
+
-
-
-
-
+
-
-
-
-
+
-
-
-
-
-
-
-
-
+
-
+
-
+
+
+
+
0
**
*
**
*
#
D
Fructose
pcDNA
CYP2J3(+)
CYP2J3(-)
CYP2J3(-)
-
+
-
-
-
-
+
-
-
-
-
+
-
-
-
-
-
-
-
-
+
-
+
-
+
+
+
+
ETA/GAPDH
(Rel Intensity)
0
2
1
4
3
5
ETA
GAPDH
*
**
#
Fructose
pcDNA
CYP2J3(+)
-
+
-
-
-
-
+
-
-
-
-
+
-
-
-
-
-
-
-
-
+
-
+
-
+
+
+
+
ET-1/GAPDH
(Rel Intensity)
0
2
1
4
3
5
ET-1
GAPDH
*
**
#
A
B
FIG. 2. Expression of IRS-1, eNOS, ET-1, and ETA-R in fructose-treated rats. Fructose administration to rats resulted in significantly decreased IRS-1
proteinlevelsinliver(A)andskeletalmuscle(B)andsignificantlydecreasedeNOSproteinlevelsinaorta(C).Theseeffectswereinhibitedbyinjection
of CYP2J3?, but not by injection of CYP2J3?. Representative Western blots are shown above graphs summarizing densitometric quantification. *P <
0.05 versus N ? pcDNA; #P < 0.05 versus F ? pcDNA; **P < 0.05 versus F ? CYP2J3?; n ? 8 per group. Levels of ET-1 (D) and ETA-R (E) transcripts
relative to glyceraldehyde-3-phosphate dehydrogenase were assessed by RT-PCR in aortic tissue samples from three rats from each treatment group
at week 5 of the study. Representative RT-PCR of ET-1 or ETA-R mRNA expression and corresponding densitometric quantification of three
experiments are shown. Values shown are mean ? SEM. *P < 0.05 versus N ? pcDNA3.1; #P < 0.05 versus F ? pcDNA3.1; **P < 0.05 versus F ?
pcDNA-CYP2J3?. Effects of CYP2J3 gene delivery on rat insulin receptor signaling and eNOS expression. Fructose administration to rats resulted in
significantly decreased eNOS protein levels in aorta (C) and decreased IRS-1 protein levels in liver (D) and skeletal muscle (E). These effects were
inhibited by injection of CYP2J3?, but not by injection of CYP2J3?. Representative Western blots are shown above graphs summarizing densitometric
quantification. *P < 0.05 versus N ? pcDNA; #P < 0.05 versus F ? pcDNA; **P < 0.05 versus F ? CYP2J3?; n ? 8 per group.
CYP2J3 ATTENUATES INSULIN RESISTANCE
1000DIABETES, VOL. 59, APRIL 2010diabetes.diabetesjournals.org

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protein in db/db mice was demonstrated by the nearly
fivefold increase in urinary 14,15-DHET levels in both
C57BL/6 and db/db mice injected with CYP2J3?compared
with those injected with the empty pcDNA vector (Fig.
3B).
The diabetic phenotype of db/db mice (injected with the
empty pcDNA vector) was confirmed by significantly
higher levels of serum glucose, serum insulin, insulin
resistance (HOMA-IR), serum triglycerides, serum choles-
terol, and urine volume in these animals compared with
those in C57BL/6 mice (all P ? 0.05; Table 3 and supple-
mentary Table 4). Injection of CYP2J3?significantly re-
duced insulin resistance (HOMA-IR) and urine volume in
db/db mice, whereas injection of the empty pcDNA vector
did not (Table 3 and supplementary Table 4). Although not
all parameters of the diabetic phenotype in db/db mice
CYP2J3
β-actin
db/db + pcDNA db/db + CYP2J3(+)
AKHL
LH
A
0
1
2
3
4
5
A
H
L
K
*
* * *
CYP2J3/β-actin
(Relative Intensity)
db/db + pcDNA
db/db + CYP2J3(+)
B
C
D
CYP2J3(+)
pcDNA
-
-
-
-
-
+
+
+
+
0
10
30
20
50
40
Urinary 14,15-DHET
(ng/ml)
-
--
C57BL/6
db/db
*
*
Series1
C57BL/6+pcDNA3.1
C57BL/6+CYP2J3(+)
db/db
db/db+pcDNA
db/db+CYP2J3(+)
db/db
db/db+pcDNA3.1
db/db+pcDNA-2J3(+)
0
10
30
50
Blood Glucose (mM)
20
40
0120 6030
Time (min)
C57BL/6
C57BL/6+pcDNA
*
*
*
*
#
#
#
#
C57BL/6
C57BL/6+pcDNA3.1
C57BL/6+pcDNA
C57BL/6+CYP2J3(+)
db/db
db/db+pcDNA
db/db+CYP2J3(+)
db/db
db/db+pcDNA3.1
db/db+pcDNA-2J3(+)
0
2
4
Plasma Insulin (ng/ml)
1
3
0120 60 30
Time (min)
C57BL/6
*
*
*
*
#
#
#
#
FIG. 3. Effects of CYP2J3 gene delivery on CYP2J3 protein expression, urinary 14,15-DHET levels, and glucose tolerance in db/db mice. A: Levels
of CYP2J3 protein were increased in the aorta (A), heart (H), liver (L), and kidney (K) in diabetic db/db mice 2 weeks after injection with
CYP2J3?compared with levels observed after injection with the empty pcDNA vector. B: Urinary 14,15-DHET levels were increased in C57BL/6
and db/db mice injected with CYP2J3?compared with those injected with the empty pcDNA vector. *P < 0.05 versus control and pcDNA-treated
mice of corresponding genotype; n ? 8 per group. Blood glucose (C) and plasma insulin (D) levels before and up to 120 min after a single glucose
challenge were elevated in db/db mice and decreased by injection with CYP2J3?. No such reduction was observed in db/db mice injected with
empty vector (pcDNA). *P < 0.05 versus C57BL/6; #P < 0.05 versus db/db ? pcDNA; n ? 8 per group.
TABLE 3
Physiological parameters determined in mice 2 weeks after injection of empty pcDNA3.1 vector or pcDNA-2J3?
VariablesC57BL/6
C57BL/6 ?
pcDNA3.1
C57BL/6 ?
p2J3?
db/db
db/db ?
pcDNA3.1
db/db ?
p2J3?
Glucose (mmol/l)
Triglycerides (mmol/l)
Insulin (mIU/l)
Fasting glucose (mmol/l)
Fasting insulin (ng/ml)
HOMA-IR
Body weight (g)
10.48 ? 0.57
1.2 ? 0.15
2.06 ? 0.35
5.54 ? 0.33
0.23 ? 0.02
0.053 ? 0.006
25.48 ? 0.61
9.78 ? 0.38
1.5 ? 0.22
2.13 ? 0.36
5.67 ? 0.26
0.22 ? 0.03
0.055 ? 0.005
25.45 ? 0.68
9.70 ? 0.60
1.43 ? 0.14
1.46 ? 0.31
5.18 ? 0.40
0.23 ? 0.01
0.052 ? 0.002
25.27 ? 0.47
21.99 ? 1.83*
2.26 ? 0.28*
7.66 ? 1.33*
17.52 ? 3.20*
2.49 ? 0.57*
2.19 ? 0.72*
45.16 ? 0.91
23.19 ? 2.51*
2.63 ? 0.00.32*
7.25 ? 0.87*
18.80 ? 2.35*
2.618 ? 0.19*
2.26 ? 0.45*
45.06 ? 0.97
23.15 ? 2.36
2.57 ? 0.37
8.25 ? 1.96
12.65 ? 1.14†
1.43 ? 0.36†
0.81 ? 0.24†
45.78 ? 1.57
Serum glucose, serum insulin, insulin resistance (HOMA-IR), and serum triglyceride levels were significantly higher in the diabetic phenotype
of db/db mice (injected with the empty pcDNA vector) compared with those in C57BL/6 mice. Injection of CYP2J3?significantly reduced
insulin resistance (HOMA-IR) in db/db mice, whereas injection of the empty pcDNA vector did not. *P ? 0.05 vs. C57BL/6 group; †P ? 0.05
vs. db/db ? pcDNA3.1 group; n ? 8 per group.
X. XU AND ASSOCIATES
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