INTERNATIONAL JOURNAL OF IMMUNOPATHOLOGY AND PHARMACOLOGY

Vol. 26, no. I, 59-73 (2013)

CDId-ASSOCIATED EXPRESSION OF NF-KB AND CARDIAC DYSFUNCTION IN

DIABETIC AND OBESE MICE
R. MADONNA, H. WU, H. SHELAT and Y-J. GENG

The University ofTexas Health Science Center at Houston and the Texas Heart Institute at
St. Luke s Episcopal Hospital, Houston, Texas, USA
Received June 29, 2012 - Accepted January 2013
The first two authors contributed equally to this study

In patients with obesity and diabetes mellitus, abnormal production of inflammatory factors may
result in cardiovascular dysfunction. In the current study, we tested the impact of CDld-mediated
innate immune responses on the expression and activation of NFKB in the hearts of adipose diabetic
(db/db) mice. Splenocytes from adult db/db and CDld-knockout mice of both genders and their wildtype, C57BL/6 and Balb/C counterparts were examined for tumor necrosis factor (TNF)-a and TNF-a
receptor type 1. The percentage of natural killer T (NKT) cells in CD3+ T cells was compared with
that in nondiabetic control mice. Despite the absence of inflammatory infiltrates, the hearts of db/db
mice showed alterations in TNF-a receptor-I and NFKB activity, including increased expression of both
the NFKB p52 and p65 subunits. In the hearts of CDld-knockout mice, p52 expression was reduced,
while p65 expression remained largely unchanged. On echocardiography, the ratio of E to A transmitral
flow velocities (an indicator of diastolic function) was significantly decreased in db/db mice after they
swam for 30 minutes. These results provide evidence for CDld-mediated NFKB activation and diastolic
dysfunction in the hearts of db/db mice. Therefore, CDld-associated abnormalities of innate immune
responses and TNF-a production in splenic tissue may contribute to NFKB activation and cardiac
dysfunction in type 2 diabetes.
and interleukin-l ~, chronic hyperglycemia creates a
proinflammatory milieu that leads to insulin resistance
and cardiovascular inflammatory complications,
such as atherosclerosis, ischemic myocardial disease,
heart failure, and dilated cardiomyopathy (2).
Researchers are increasingly recognizing that
activation of the innate immune system plays
a role in the development and progression of
insulin resistance by inducing chronic, low-grade

The concept that type 2 diabetes mellitus is a

chronic inflammatory disease is widely accepted,
owing to the growing body of evidence indicating
that chronic, low-grade inflammation serves as a
common denominator of diabetes, linking obesity,
insulin resistance, and cardiovascular complications
(1). By increasing the circulating levels of
proinflammatory cytokines such as tumor necrosis
factor (TNF)-a, monocyte chemoattractant protein-I,

Key words: obesity, diabetes mellitus, type 2, nuclear transcription factor CDld, diastolic dysfunction, low-grade
chronic inflammation
Mailing address: Yong-Jian Geng, MD, PhD,
Professor and Director, Center for Cardiovascular Biology
and Atherosclerosis Research, Cardiology Division,
Department ofIntemal Medicine, The University of Texas
Medical School at Houston, 6431 Fannin Street,
MSB 1.246, Houston, TX 77030, USA.
Tel.: +1713 5006073/713 5006569 Fax: +1 713 5006556
e-mail: yong-jian.geng@uth.trnc.edu

0394-6320 (2013)
copyright by BIOLlFE, s.a.s.
This publication and/or article is for individual use only and may not be further
reproduced without written permission from t1iecopyright holder.
Unauthorized reproduction may result in financial and other penalties

59

DISCLOSURE: ALL AUTHORS REPORT NO CONFLICTS OF

INTEREST RELEVANT TO THIS ARTICLE.

60

R. MADONNA ET AL.

inflammation (3). Although the innate immune

system provides the first line of defense against
microbial pathogens, it also plays an important role
in amplifying the inflammatory response, which
does not depend on antigenic stimulation. Both
infection-induced and sterile inflammation can lead
to chronic inflammation, a key etiological condition
in the development of insulin resistance. However,
the molecular and cellular basis of innate immune
regulation in the progression of diabetes-associated
cardiovascular diseases has not been fully elucidated.
The spleen contains a variety of innate immune
cells, predominantly natural killer (NK) and
invariant natural killer T (NKT) cells (4). The
proportion of NKT cells is considerably higher in
splenic mononuclear cells than in blood and other
lymphoid organs, including the thymus (5). NKT
cells are a heterogeneous subset of lymphocytes that
express both NK and T cell surface markers (6). In
mice, classic (type I) NKT cells express an invariant
T cell receptor containing a Va14-Ja18 chain,
whereas nonclassic (type II) NKT cells express
diverse T cell receptors (6). NKT cells recognize
a glycolipid antigen presented by CDld, a major
histocompatibility complex molecule expressed in
antigen-presenting cells such as dendritic cells and
macrophages (6). Because CDld is essential for the
development of NKT cells (6), genetic disruption of
this molecule depletes both type I and type II NKT
cells in the whole body (7).
There is a growing consensus that glucose- and
diabetes-induced activation of nuclear factor-KB
(NFKB) contributes to the proinflammatory diabetic
milieu, including heart tissue inflammation (8). The
NFKB family of transcription factors is controlled
by 2 distinct mechanisms of activation known as
the canonical (classic) pathway, which determines
the release of p50/p65 dimers to the nucleus, and
the noncanonical pathway, which causes pi 00
processing and nuclear translocation of RelB/p52
(9). The canonical pathway plays an important role
in the host's innate immune response by inducing
expression of numerous proinflammatory cytokines,
chemokines,
adhesion
molecules,
inducible
enzymes, and proangiogenic growth factors, as well
as by inhibiting apoptosis (10). The more recently
identified noncanonical pathway plays an important
role in the adaptive immune response, including

secondary lymphoid organogenesis and lymphocyte

maturation (11).
We hypothesized that the CDld-mediated
splenic inflammatory response activates the innate
immune pathway of cardiac muscle cells in a
paracrine manner, by secreting cytokines such as
TNF -a and consequently activating the canonical
NFKB pathway. We sought to determine whether
direct activation of innate immune sensors by CDldrestricted NKT cells in the spleen is associated with
expression and activation of the NFKB signaling
pathway in the hearts of obese diabetic (db/db) mice
with insulin resistance. In this report, we describe
the association of splenic inflammatory responses
with the expression of TNF-a receptor type 1 and
strong activation of NFKB, significant alteration
of canonical RelNp65, and to a lesser extent,
expression of the noncanonical RelB/p52 subunit in
the hearts of db/db mice. Our observations suggest
that the abnormal immune responses and TNF-a
production in spleen tissue plays a role in NFKB
activation, which is crucial for the development
and progression of cardiovascular dysfunction in
diabetes.
MATERIALS AND METHODS
Animals and their care

The study population comprised wild-type C57BL/6

mice (body weight: 224 g; 3 males and 3 females), wildtype Balb/C mice (body weight: 214 g; 3 males and
3 females), COld-knockout mice (body weight: 255
g; 3 males and 3 females), and Lepr" (db/db) mice on
a C57BL/6 background (homozygotes for a mutation in
the leptin receptor gene, leading to the loss of functional
leptin receptors) (12) (body weight: 765 g; 3 males and
3 females). All mice were 12-month-old adults obtained
from The Jackson Laboratories (Bar Harbor, Maine,
USA).
TablesI and II showthe physiologicaland biochemical
characteristics of db/db, C57BL/6, CDld-knockout, and
Balb/C mice. Db/db mice on a C57BL/6J background
develop hyperphagia, obesity, and insulin resistance,with
severe depletion of the insulin-producing beta-cells of the
pancreatic islets (12). The diabetic phenotype of these
mice, however,is less severe than that of db/db mice on a
KsJ background(13). The latter are more severely insulin
resistant but less hyperglycemic than db/db mice on a
C57BL/6J background (13). Moreover, db/db mice on a
C57BL/6J background develop higher plasma fatty acid

Int. J. Immunopathol. PharmacoI.

levels than do db/db mice on a KsJ background.

All mice were specific-pathogen free and kept in
a temperature-controlled environment in a ventilated
rack with a 12:12-hour lightdark cycle. Food and water
were given ad libitum. All procedures were approved
by the Institutional Ethics Committee (University of
Texas Health Science Center at Houston) for animal
research. The investigations conformed to the Principles
of Laboratory Animal Care formulated by the National
Society for Medical Research and the Guide for the Care
and Use of Laboratory Animals published by the National
Institutes of Health (USA).

Materials
All chemicals were purchased from Sigma-Aldrich (St.
Louis, Missouri, USA) unless otherwise specified. Type-I
collagenase was obtained from Worthington Biochemical
Corporation (Lakewood, New Jersey, USA).
Blood collection and biochemical assays
Blood was drawn via a cardiac puncture, collected into
nonheparinized tubes, allowed to clot at room temperature
for 1 hour, and centrifuged for 20 minutes at 2,000 x g.
The serum was removed and stored at -20 DC until assays
were performed. Levels of glucose, triglyceride, and
the total, high-density lipoprotein (HDL], low-density
lipoprotein (LDL), and very-low-density lipoprotein
(VLDL) cholesterol were measured by standard clinical
chemistry laboratory methods (Equine Laboratories,
Houston, Texas, USA).
Flow cytometry
After being anesthetized with an intraperitoneal
injection of 65 mg/kg ketamine and 13 mg/mL xylazine
in sterile normal saline, the wild-type C57BL/6 and db/db/
C57BL/6 mice were euthanized. The spleens of male db/
db mice and age- and sex-matched C57BL/6J control mice
were removed, mashed by the plunger of the syringe, and
passed through a 70 IlM cell strainer. After red-blood-cell
removal, spleen cells were plated in RPMI 1640 culture
medium supplemented with 20% fetal bovine serum
(Gibco, Invitrogen; Life Technologies, Grand Island, New
York, USA), penicillin (100 U/mL), and streptomycin
sulfate (100 mg/mL). For flow cytometry analysis and cell
sorting, spleen cells (1x 106) were incubated for 30 minutes
at 4DC with 10 ul, ofphycoerythrin-conjugated mouse antiCDld antibody (Santa Cruz Biotechnology, Santa Cruz,
California, USA). An antibody specific for the FcyRIII/II
receptor (BD Biosciences, San Jose, California, USA) was
used to inhibit nonspecific staining. Cells were analyzed
with a BS LSRII flow cytometer (BD Biosciences). For
each sample, 30,000 events were acquired and analyzed
with CellQuest software (BD Biosciences). Subsequently,

61

CDld+ splenocytes were sorted from CDld- splenocytes

with a fluorescence-activated cell sorter (BS LSRII flow
cytometer sorter; BD Biosciences) and used for further
analyses.

RNA Analysis by Quantitative Real-time Polymerase

Chain Reaction (qRT-PCR)
Total RNA was isolated with a Qiagen RNA isolation
kit. First-strand eDNA synthesis was performed with an
iScript eDNA Synthesis Kit (Bio-Rad Laboratories,
Hercules, California, USA) according to the manufacturer's
protocol. The following qRT-PCR primers, designed
with Oligof'erfect" Designer software (Invitrogen),
were used: mCDl(5'-AAGCTGGTCCCGCACAGA-3',
3' -GCTGATGGTGGCTGAGTCATT-5'),
TNF-a
(5' -TCGTAGCAAACCACCAAGTG-3',
3' -AGATAGCAAATCGGCTGACG-5'),
TNF-a
receptor! (5' -CCATCTTCGGTCCTAGTAACTG-3',
3'CAGGTTCATCTTGGAAAGCAC-5 '),
TNFa
receptor2
(5' -GATGCCAAGGTGCCTCATG-3',
3' GAGCTGCTACAG ACGTTCACG-5'),
brain
natriuretic
peptide
(BNP)
(5' -CTGAAGGTGCTGTCCCAGAT-3',
3' -CCTTGGTCCTTCAAGAGCTG-3 '). All qRT-PCR
reactions were performed in triplicate by using a MyiQTM
Single-Color Real-Time PCR detection system (BioRad). A melting curve was generated at the end of every
run to ensure product uniformity. Relative values were
obtained by normalizing CT values of the tested genes
in comparison with CT values of the housekeeping gene
RNA GAPDH, using the ~ CT method.
Tissuepreparation and histological analyses
After anesthesia was induced, the hearts from wildtype C57BL/6 and db/db/C57BL/6 mice were perfusionfixed with 4% paraformaldehyde and 5% sucrose in
phosphate-buffered saline (PBS) for 10 min, then removed
from the chest, embedded in optimal-cutting-temperature
(OCT) compound, frozen on dry ice, and stored at -70 DC
until sectioning. Serial 7-llm-thick sections were obtained
by using a sliding cryotome. For immunofluorescence
staining, these sections were permeabilized, blocked for
30 min in PBS containing 1% bovine serum albumin, and
incubated for 1 h at 4 DC with primary antibody against
rabbit sarcomeric n-actinin cross-reacting with mouse.
After being washed with PBS, the heart sections were
incubated with fluorescein isothiocyanate-conjugated
antirabbit secondary antibody. Nonimmune IgG was
used as the isotype control (Becton, Dickinson and Co.,
Franklin Lakes, New Jersey, USA). The slides were
washed, mounted, and viewed through a fluorescence
microscope.
Heart sections were also prepared with hematoxylin

62

R. MADONNA ET AL.

and eosin (H&E) stain for structural analysis by light

microscopy, with Sirius red stain to assess the presence
and extent of fibrosis and with Oil Red a stain to
identify intramyocardial lipid deposition. Images were
obtained with a digital camera and analyzed by using the
Metamorph Image System (Molecular Devices Corp.,
Downingtown, Pennsylvania, USA).

Electrophoretic Mobility Shift Assays (EMSAs)

Nuclear proteins from wild-type C57BL/6 and db/dbl
C57BL/6 mice and from wild-type Balb/C and CDldknockout mice were extracted from snap-frozen hearts as
previously described (14). Briefly, tissues were harvested and
homogenized with an Ultra-Turrax T25-tissue homogenizer
(Janke and Kunkel) in a low-salt solution (0.6% Nonidet
P-40; 150mMNaCl; 10 mM HEPES, pH 7.9; 1 mMEDTA;
0.5 mM phenylmethylsulfonyl fluoride), and centrifuged for
30 seconds at 2,000 rpm. The supernatant was incubated
on ice for 5 minutes and then centrifuged at 5,000 rpm for
5 min. The nuclei were resuspended in a high-salt solution
(25% glycerol; 20 mM HEPES, pH 7.9; 420 mM NaCl;
1.2 mM MgC12; 0.2 mM EDTA; 0.5 mM dithiothreitol; 0.5
mM phenylmethylsulfonyl fluoride; 2 mM benzarnidine;
5 g/mL each aprotinin, leupeptin, and pepstatin). Protein
concentrations were determined by Bio-Rad Laboratories
Protein Assay. Double-stranded NFJeB oligonucleotides (5'AGT TGA GGG GAC TIT CCC AGG C-3' and 5'-CCT
GGG AAA GTC CCC TCA ACT-3') (promega, Madison,
Wisconsin, USA) were labeled with [y_32P]ATP. Binding
reactions containing 10 ug of crude nuclear extract were
performed by using an EMSA core system (Promega)
according to the manufacturer's protocol. For supershift
assays, I ~L of goat monoclonal anti-p65 antibody (Santa
Cruz Biotechnology) was added into the reaction tube, with
a final volume of I0 ~L, containing I0 ug of crude nuclear
extract.
Immunoblottingfor p65 andpI 00/52 NF-KB subunits
Total proteins from wild-type C57BL/6 and db/dbl
C57BL/6 mice and from wild-type Balb/C and CDldknockout mice were extracted from heart, spleen, lymph
node, and heparinized peripheral blood samples snapfrozen in liquid nitrogen. At 4C, the frozen samples
were ground into a fine powder and homogenized by a
polytron in cold lysis buffer (50 mM HEPES, pH 7.5; 150
mM NaCl; 10 mM NaPP; 2 mM Na 3V04 ; I mM MgCI 2;
I mM CaCI 2; 10 mM NaF; 2 mM EDTA; 2 mM PMSF;
5 ug/ml, leupeptin; 1% NP-40; 10% glycerol). They
were then centrifuged at 25,000 rpm at 4C for 30 min
to remove insoluble material, and the supernatant was
used as a sample. An aliquot of the nuclear extracts used
for EMSA and cytosolic or total extracts were loaded
on 5-10% SDS-polyacrylamide for gel electrophoresis

under reducing conditions (14) and were electroblotted

onto polyvinylidene fluoride membranes (Immobilon-P+;
Millipore, Bedford, Massachusetts, USA). Each
membrane was blocked in 3% goat serum for I h and
incubated overnight at 4C with the following primary
antibodies: I) polyclonal anti-rabbit p65 (Santa Cruz
Biotechnology); 2) polyclonal anti-rabbit plO0/52 (Cell
Signaling Technology, Inc., Danvers, Massachusetts,
USA); 2) anti-B-actin (Sigma-Aldrich); 3) anti-lamin B
(Santa Cruz Biotechnology). The blots were developed
using a SuperSignal West Pico Chemiluminescent
Substrate kit (Pierce Biotechnology, Inc., Rockford,
Illinois, USA). The intensity of each immunoreactive
protein band was measured by densitometry.

Swimming exercise and echocardiography

Sex- and age-matched db/db and wild-type (C57) mice
were subjected to a 30-minute swimming session followed
by 5 minutes ofrest. Constant monitoring ensured the safety
of the mice and prevented them from floating or holding
their breath under water. Before swimming (baseline)
and after completion of the swimming-rest program, the
mice underwent transthoracic echocardiography. They
were anesthetized with ketamine (100 mg/Kg) and placed
in the supine position. Two-dimensional and M-mode
echocardiographic images were recorded and analyzed
by a Vevo 770 echocardiograph equipped with a 40MHz ultrasonic linear probe (Visual Sonics, Toronto,
Canada). Two-dimensional transverse left ventricular
(LV) imaging was used to position the probe just distal
to the mitral valve leaflets, and M-mode images were
then captured in the short and long axes. Three loops of
M-mode data were captured from each animal and stored
on digital disk until analysis. Each image loop provided
5 to 12 heart cycles; data were averaged from at least 5
cycles per loop. Left ventricular internal systolic (LVIDs)
and diastolic (LVIDd) diameters were measured, as were
diastolic anterior and posterior wall thickness, according
to the American Society of Echocardiography's leadingedge technique. These parameters allowed LV fractional
shortening (% FS), a measure of systolic function, to be
determined by the equation: % FS = [(LVIDd - LVIDs)1
LVIDd] x 100%. Using Teichholz's formula, end-diastolic
volume [EDV: 7.0/(2.4 + LVIDd) x LVID s3], end-systolic
volume [ESV: 7.0/(2.4 + LVIDs) x LVIDs3], and ejection
fraction [%: LVEDV - LVESV/LVEDV) x 100] were
calculated. Left ventricular mass was calculated as 1.055
x [(LVIDd + PW + AW)3 0 (LVIDd)3], where PW and
AW were the thickness of the posterior and anterior LV
walls, respectively.
Statistical analysis
All results are presented as mean values standard

Int. J. Immunopathol. Pharmacol.

deviation (SD). Two-group comparisons were performed

by using Student's r-test for unpaired values. Multiplegroup comparisons were made by using analysis of
variance (ANOVA). P values of less than 0.05 were
considered significant. Statistical analyses were carried out
with SPSS software, version 15.0 (SPSS, Inc., Chicago ,
Illinois , USA).

RESULTS

Obesity, lipid profiles, and CDld expression in db/

db mice
As expected, 12-month-old db/db mice fed normal
chow gained significantly in body weight, with
marked elevation of blood lipids and glucose levels.
At the same age, db/db mice were much heavier
than nondiabetic C57 control mice (P<O .OI; Table

C57

(J)
(J)

I). These 2 groups also had a modest, but significant

difference in heart weight and heart weight/body
weight ratio (P<O.05) . The blood levels of glucose,
total cholesterol, LDL and VLDL cholesterol, and
triglycerides were significantly increased in the db/
db mice versus nondiabetic control mice (P<O.O I;
P<O.OI; P<O.OI; P<O.05; or P<O.OI, respectively ;
n=3 mice per each group; Table I). Thus, compared
to control C57 mice, db/db mice developed severe
obesity, hyperlipidemia , and hyperglycemia.
Expression of CD Id in the spleens of C57BL/6J
and db/db mice was analyzed by flow cytometry
with phycoerythrin-conjugated anti-CD Id antibody.
Higher numbers of spleen cells displayed CDld
expression in db/db mice than in sex- and agematched C57 control mice [56.9 2.3% (n=3) vs

db/db
~ b

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J

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55.2%

D...

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80

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63

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CD1d-PE

0
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20
0

C57

D
C57

db/db

.....

db/db

*
1.6

1.4
> 0:::: 1.2
~ E
1.0
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.-~ ......
roo 0.8
Qi U
0.6
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0.4
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0

CD1d

GAPDH

QiZ

C57

db/db

Fig. 1. CDld expression in spleens from C57BL/6J and db/db mice. Panels A, B) Flow cytometry analysis of CDld
exp ression in spleen cells from male db/db mice, compared with the respective sex- and age-matched (12-month-old) wildtype C57BL/6J control mice. In Panel A, the scatter plots are representative of 3 independent experiments. Controls for
flow cytometry included an IgG isotype control antibody. In Panel B, the columns and bars represent the mean percentage
standard deviation (SD) ofCDld-positive cells (*P<O.05, db/db mice versus C57BL/6J control mice) . Panels C, D)
Quantitative real-time polymerase chain reaction (qRT-PCR) assessment ofCDld mRNA exp ression in spleen cells from
male db/db mice, compared with their resp ective sex- and age-matched (12-month-old) wild-type C57BL/6J control mice.
The columns and bars represent the mean values SD from 3 independent experiments assessing mRNA expression after
normalization with GAPDH. In Panel C. agarose gel [electrophoresis?] ofqRT-PCR [products?] is representative of3
independent experiments (*P<O.05, db/db mice versus C57BL/6J control mice).

64

R. MADONNA ET AL.

C57

db/db
TNF a

GAPDH

B
7

z
0::: 5

0
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~o:::
~ E 4

**

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> ....

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db/db

C57

db/db

Fig. 2. Expression oftumor necrosis factor (TNF)-a and TNF-a receptorl in spleens and hearts from C57BLl6J and db/db
mice. Panels A, B) Quantitative real-time polymerase chain reaction (qRT-PCR) assessment ofTNF-a mRNA expression
in spleen cells from male db/db mice, compared with the respective sex- and age-matched (l2-month-old) wild-type
C57BLl6J control mice. In Panel A, agarose gel electrophoresis ofqRT-PCR products is representative of 3 independent
experiments. Panel B, the columns and bars represent the mean values standard deviation (SD) from 3 experiments
assessing mRNA expression after normalization with GAPDH (**P<O.Ol, db/db mice versus C57BLl6J control mice).
Panel C) qRT-PCR assessment ofTNF-a receptor1 mRNA expression in hearts from male db/db mice, compared with the
respective sex- and age-matched (l2-month-old) wild-type C57BLl6J control mice. The columns and bars represent the
mean values SD from 3 experiments assessing mRNA expression after normalization with GAPDH (**P<O.Ol, db/db
mice versus C57BLl6J control mice).

42.85.8% (n=3); P<O.05, db/db vs. C57BL/6 mice]

(Fig. 1 A, B). Quantitative RT-PCR showed that
CD1d mRNA expression increased in splenocytes
from db/db mice compared with C57BL/6 control
mice after normalization with GAPDH mRNA levels
(Fig. 1 C,D). Thus, CDI expression is increased in
the splenocytes of db/db mice.

receptor-l (Fig. 2C), but not receptor 2 mRNA (Fig.

2D), was significantly increased in the db/db mouse
hearts, corresponding to the elevation of TNF-a
mRNA in the splenocytes of db/db mice. On qRT-PCR,
expression ofTNF-a receptor-l was 4.9-fold higher in
the hearts of db/db mice than those ofC57BL/6J mice
(P<O.Ol db/db vs C57 mice) (Fig. 2C).

Increased splenocytic TNF-o. and cardiac TNF-o.

receptor-l mRNA expression in db/db mice
TNF-a mRNA in the spleens ofC57BL/6J and db/
db mice was measured by using qRT-PCR. As shown
in Fig. 2 A,B, a 6-fold increase in the levels ofTNF-a
mRNA was seen in the spleen cells of db/db versus
C57BL/6 mice. Conversely, expression of TNF-a

Differential p65 subunit expression and activities in

hearts ofdb/db and CDld-knockout mice
The nuclear transcription factor NFKB binds to
the promoters that are critical for transcription of
a variety of proinflammatory genes. We analyzed
the expression and activities of NFKB in the hearts
of db/db and C57 mice. Levels of the NFKB p65

Int. J. Immunopathol. Pharmacol.

C57
M

db/db
F

Balb/C

CD1d-l -

DC
..

65

-I

p65

c
Ant i-p65

C57

M
F
+ - + -

[e65

Lamin B

I Lamin B

db/db

M
+

F
- + -

123456789
-

supershift

shift , p65 NFKB complex

Fig. 3. NFKB expression and activity in hearts from db/db and nondiabetic control mice. Panel A) Levels of the p65
subunit ofNFKB protein in nuclear protein extracts isolatedfrom 12-month-old db/db male (M) andfemale (F) mice and
sex-matched nondiabetic C57BL/6 control mice. Panel B) Levels of the p65 subunit ofNFKB protein in cytosolic and
nuclear protein extracts isolatedfrom 12-month-old male CD1d-knockout and sex-age matched Balb/C control mice. The
blots are representative of 3 independent experiments. Similar results were obtainedfrom female mice. Panel C) NFKB
activity was determined by an electrophoretic mobility shift assay, performed by mixing a 32P-labeled oligonucleotide
that codes for the consensus sequence ofNFKB binding promoter with nuclear proteins from the hearts ofmale (M) and
female (F) db/db mice and sex- and age-matched (l2-month-old) C57BL/6 control mice (3 mice per group). Specificity of
NFKB binding activity was evaluated by cold oligonucleotide analysis and supershifting with anti- NFKB antibody. The
electrophoretic run with nuclear protein extracts from C57BL/6 control hearts is shown in lanes 1-4. The electrophoretic
run with nuclear protein extracts from db/db mice is shown in lanes 5-8. The autoradiogram is representative 3 separate
gel shift experiments for NFKB. Lane 9 shows the electrophoretic runs with protein extracts omitted or with cardiac
nuclear extracts incubated with a cold probe.

subunit were determined by Western blot analysis in

db/db and CDld-knockout mice and their wild-type
counterparts (C57BL/6 and Balb/C, respectively).
Stronger bands of p65 proteins were found in the
cardiac nuclear extracts of db/db mice than of C57
control mice, especially in female animals (Fig.
3A). Also, p65 was found in the hearts of CD1d-null

and Balb/C mice (Fig. 3B). However, the nuclear

expression of the NFKB p65 subunit in the hearts of
CD1d-knockout mice appeared to be lower than that
of the wild-type controls (Fig. 3B).
To determine the NFKB activities, we conducted
a gel shift assay with 32p end-labeled NF-kB
oligonucleotides, using the nuclear proteins isolated

66

R. MADONNA ET AL.

Cytosol

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B-actin

Cytosol
'):

cl (]

34-

-I

()

...

55 -

Total

se

,.

72-

p52

55
43

Total

LO

0..

C57

db/db

Balb/C CD1d-l -

Fig. 4. RelB/p52 expression and activity in hearts and spleens from CDId-knockout and db/db mice. Levels of the
endogenous p l 00 precursor and p52 active subunit ofNF-kB complex in total and cytosolic heart protein extracts isolated
from male db/db mice (Panel A) and male CDId knock-out mice (Panel C), compared with the respective background
sex- and age-matched (l2-month-old) wild-type C57BL/6 and Balb/C control mice. Panel B) Levels ofthe endogenous
plOOprecursor and p52 active subunit ofNF-kB complex in total spleen protein extracts isolatedfrom male db/db mice
compared with sex- and age-matched (l2-month-old) wild-type C57BL/6 control mice. The blots are representative of3
independent experiments. The results ofscanning densitometry (3 mice per group) are expressed as arbitrary units. The
columns and bars represent the mean values standard deviation (**P<O.OI, db/db mice versus control mice).

from snap-frozen hearts. NFKB binding actrvmes

were much stronger in the nuclear extracts from ageand sex-matched hearts of db/db mice than from
the wild-type controls (Fig. 3C). The specificity
of the NFKB-DNA protein complex formation was
verified by competition with cold oligonucleotides
and by addition of anti-p65 NFKB antibody, which
super-shifted the bands of NFKB/DNA complexes
(Fig. 3C, lanes 1,3,5, 7). Antibody to p65/RelA was
capable of super-shifting a portion of the protein
binding to the nuclear DNA, indicating p65 subunit
involvement in activation of NFKB in the db/db
hearts. The binding activity with the radioactive

oligonucleotide probe was abolished in the absence

of protein extracts (Fig. 3C, lane 9) indicating the
dependence on nuclear protein factor binding for the
shifted radioactive bands.
CD1d association with NF-kB precursor expression
andprocessing in hearts
The NFKB p65 subunit was highly detectable
in cytosolic extracts from CDld-knockout mice
(Fig. 3B), indicating lack of cytosolic-nuclear
redistribution of p65 and, therefore, lack of
activation of canonical NFKB in the absence of
CD 1d. When whole heart extracts were analyzed by

Int. J. Immunopathol. Pharmacol.

Balb/C

db/db

C57

Balb/ C

CD1-1

11 11 11 11

TC

COl -I-

II

~ g i b"'gi
<? .<f ~ <?.<f ~

blood
TCTC

67

TC

130 -

130
96

plOD

72
55

p5 2

43

plOD

96

72 55

p52

43
34

34

_~

I~-actin

~ - actin

Fig. 5. RelBlp52 expression and activity in lymph nodes, spleen and bloodfrom CDId-knockout and BalblC control mice.
Panel A) Levels ofpIOOlp52 expression in total and cytosolic protein extracts isolated from blood of male dbldb mice
and CDI d knock-out mice compared with sex- and age-matched wild-type BalblC and C57BL/6 controls mice. Panel B)
Levels of endogenous pI 00 precursor and p52 active subunit ofNF-KB complex in total protein extracts isolatedfrom
spleen, lymph nodes and blood of male CDI d knock-out mice comp ared with sex- and age-matched wild-typ e BalblC
controls. Blots are representative ofthree independent experiments.

immunoblotting for processing of the pi 00 precursor

into the p52 active subunit, levels ofp52 in the hearts
of db/db mice and nondiabetic C57BL/6 control mice
reflected p65 expression and NF-kB binding activity,
although to a lesser extent than in the other group
of mice (Fig. 4A). A marked reduction in RelB/p52
expression was observed in the hearts of CD 1dknockout mice versus Balb/C control mice (Fig. 4C).
The spleen (Fig. 4B) and blood (Fig. 5A) showed
marked expression of RelB/p52 in db/db mice
compared with nondiabetic C57BL/6 control mice.
However, in the spleen and blood of CD1d-knockout
mice, no significant levels of p52 were detected (Fig.
5B), indicating lack of activation of noncanonical
NF-KB in the absence ofCDld.

Cardiomyocyte lipid accumulation and global

morphology ofdb/db hearts
Insulin resistance is frequently associated with
lipid deposition in nonadipose tissue. The hearts
of db/db mice showed large quantities of lipid
accumulation within the ventricles, as visualized with
Oil red a staining (Fig. 6A), along with increased
depots in the fat pads around the ventricles. Few lipid
stains were found in the hearts of C57BL/6 mice
(Fig. 6E). Although lipid accumulation was seen in

db/db mice, global morphology and cardiomyocyte

structure remained largely unchanged. In db/db
mice, no cardiac hypertrophy was apparent on H&E
staining (Fig. 6B), and no fibrosis was observed
on sirius red staining (Fig. 6D) as compared with
C57BL/6 mice (Fig. 6F, H). Immunofluorescence
staining for a-sarcomeric actinin (a crucial Z-disc
protein that cross-links sarcomeric actin) revealed
that the heart tissue had sarcomeric integrity without
microscopically detectable anomalies (Fig. 6C); the
myocytes showed patterns of striated myofibrils
similar to those of the control C57 mice (Fig. 6G).
Changes in cardiac performance in db/db mice
undergoing stress testing
M-mode measurements of LV dimensions
and indexes of systolic heart function showed
no significant difference between the db/db and
nondiabetic control mice at baseline and after exercise
(Table III; n=6). On Doppler flow analysis, the ratio
of E to A transmitral flow velocities (an indicator of
diastolic function) was significantly decreased in
db/db mice after exercise, indicating impaired LV
relaxation (Fig. 7). The exercise-induced increase in
fractional shortening was not significantly different
in db/db versus nondiabetic control mice (Table

68

R. MADONNA ET AL.

ORO

H&E

FITC-sarc a-actinin

Sirius red

Fig. 6. Histological analyses of heart sections from nondiabetic controls and db/db mice. Representative Oil Red a
stained cross-sections of left ventricular myocardium from male db/db (A) and sex- and age-matched (l2-month-old)
C57BL/6 control mice (E), showing accumulation oflipids in db/db cardiac myocytes. Hearts were harvested, embedded
in optimal-cutting-temperature compound, and counterstained with hematoxylin and eosin (H&E) (3 hearts per group).
Similar results were observed in female mice. Panels Band F: Representative H&E-stained cross-sections of left
ventricular myocardium from male db/db mice (B) and sex- and age-matched (l2-month-old) C57BL/6 control mice
(F). Similar results were observed in female mice. Panels C and G: Representative immunofluorescence-stained crosssections ofleft ventricular myocardiumfrom male db/db (C) and sex- and age-matched (l2-month-old) C57BL/6 mice (G)
stained for sarcomeric a-actin in. Similar results were observed in female mice. Panels D and H) Representative Sirius
red-stained cross-sections ofleft ventricular myocardium from male db/db (D) and sex- and age-matched (l2-month-old)
C57BL/6 mice (H). Similar results were observed infemale mice. Magnification: LO>.

Ill). Of note, there was no difference in the effect of

exercise on heart rate between db/db and nondiabetic
control mice (Table Ill), indicating a similar cardiac
compensatory response to stress by adrenergic
stimulation in the 2 groups.
Expression of BNP rnRNA, a highly sensitive
indicator of cardiac dysfunction in diabetic mice
(15) and in humans (16), was significantly higher in
db/db mice [males, 1.58O.7 pg/mL (n=4); females,
1.66OA pg/mL (n=4)] versus nondiabetic control
mice [males, O.73O.3 pg/mL; females, O.97OA
pg/mL]. Thus, increased BNP mRNA expression
appeared to change in parallel with alterations in
cardiac diastolic function in db/db mice.

DISCUSSION
An association between low-grade chronic
inflammation and cardiovascular complications in
insulin resistance and type 2 diabetes is increasingly
being recognized (l). However, the molecular and
cellular basis of immune dysregulation leading
to low-grade chronic inflammation and cardiac
malfunction in type 2 diabetes has not been fully
elucidated. Our current study provides experimental
evidence that a heightened innate immune response
occurs through a CD 1d-mediated mechanism .
In the spleen, CD 1d-restricted, lipid-responding
NKT cells result in a variety of immune reactions,

lot . J . Immuoopathol. Phar macol.

db /db
pre-swimming

o
~-r
- E
III

0-

(1)-

>

69
db /db
post-swimming

~-l
o

III

0-

-(1)E

>

Fig. 7. Echocardiographic index ofdiastolicfu nction in nondiabetic controls and db/db mice. Panels A, B) Representative
Doppler flow recordings fro m db/db mice at baseline (A) and after swimming (B), showing decreased peak E-wave
heights in db/db mice and impaired left ventricular relaxation after exercise. Panel C) The E/A ratio (measured as the
ratio between the peak height ofthe mitral flo w E-wave and that of the A-wave on Doppler flo w recording) in db/db and
C57 mice at baseline and after swimming. Each dot indicates the mean value derived fr om at least 3 measurements in
each individual mouse. The lines indicate mean values in each group ofmice (n=7).

including induction ofT helper-derived inflammatory

cytokines (17), suggesting that these cells have
complex roles in immune dysregulation and
inflammation. In this study, we hypothesized a link
between systemic low-grade chronic inflammation
and activation of selective elements of the innate
immune system, such as CDld-restricted NKT cells
in obese diabetic (db/db) mice. We demonstrated
that spleen inflammatory responses characterized
by activation of CDld-restricted NKT cells are
associated with expression and activation of NF-KB
in the hearts of adipose db/db mice. In our study,
spleen tissues of db/db mice displayed high levels
of CD1+ NKT cells, particularly in younger mice.

It is likely that activated CD1d-restricted NKT cells

produce high levels of proinflammatory cytokines,

such as TNFa and interleukin-l , which circulate to
the heart and cause cardiac malfunction (Fig. 8).
However, there is no direct evidence that CD1dassociated NKT cell activation contributes to cardiac
.dysfunction. Miyazaki and colleagues recently
reported that the population of NKT cells may
decline in mice fed a high-fat diet (108). Therefore,
high-fat feeding may have different biological
effects on CDld-mediated NKT cell function than
the endogenous hyperlipidemia developed in db/db
mice.
CDld is known to playa key role in presenting

R. MADONNA ET AL.

70

NK-T Cell

Obese diabetic (db/db) m ice

with glyco-/ipid an tig ens

Cardiac
myocyte

Dendritic Cell

Dysfunction

(jJ

TCR

Glycolipid
CD1d

Fig. 8. CDId-associated expression of NFKB and cardiac dysfunction in type 2 diabetes. In type 2 diabetes, TNF-a
production in splenic tissue may contribute to NFKB activation and cardiac dysfunction. NKT: natural killer T; TNF:
tumor necrosis factor; TNFR: tumor necrosis factor receptor; TCR: T-cell receptor.

glyco-lipid antigen molecules to certain T subsets,

particularly NKT cells (19). Conceivably, lipid
accumulation in the organs or tissues of obese
diabetic mice, including the heart, may facilitate the
generation of denatured, chemically modified lipid
molecules, which act as auto-antigens recognized by
CDld-restricted NKT cells (20). Therefore, because
CD1d plays a key role in lipid antigen presentation,
this molecule can facilitate innate immune
responses involving NKT cells, thus participating
in the inflammatory responses In obese db/db
mice. Our observations suggest that spleen CDldrestricted NKT cells playa crucial role in activating
inflammatory responses and NFKB signaling in the
hearts of such mice. This activation is crucial for
the development and progression of cardiovascular
dysfunction in diabetes.
Another interesting finding in this study concerns
CDld-dependent expression and activation of NFkB in the myocardium. Our data clearly show that
heart tissues of db/db mice express high levels
of TNF-n receptor type 1 and NFKB, paralleled
by lipid accumulation and diastolic dysfunction.
Furthermore, the heart, spleen, and blood of CD1dknockout mice, which lack mature NKT cells
systemically, display no activation ofthe p65 subunit
and have markedly reduced RelB/p52 expression,
indicating a compromised capacity for NFKBmediated inflammation in the absence of CD1d.

Recent studies have emphasized the existence

of multiple facets of NFKB in the heart, suggesting
that the timing, duration of activation, and cellular
context may explain mechanistically differential
outcomes of cardiac NFKB signaling (21). Whereas
NFKB is cardioprotective during acute hypoxia
and reperfusion injury, prolonged activation of
NFKB appears to promote heart failure by eliciting
signals that trigger chronic inflammation through
enhanced elaboration of cytokines - including
TNF-n, interleukin-I, and interleukin-6 - leading to
endoplasmic reticular stress and cell death (22).
A novel finding of our study is dysregulation of
the noncanonical NFKB activation pathway, with
significant changes in expression of the plOO/p52
protein complex. Our finding that the cytosolic p52
subunit was increased in the hearts and peripheral
blood of db/db versus nondiabetic mice indicates
a marked processing of the pi 00 precursor into
the p52 active subunit and supports activation of
the noncanonical NFKB pathway in the presence
of diabetes. Unlike canonical NFKB signaling,
noncanonical NFKB signaling in the heart has not
been widely studied (21). In other cell systems, the
major ligands of the noncanonical pathway include
lymphotoxin-B, B-cell activating factor, and the
CD40 ligand (23). Activation of these respective
receptors results in phosphorylation of plOO at
Ser866 and Ser870 and subsequent processing,

lot. J. ImmuoopathoL Pharmacol,

resulting in active p52 (24). In previous studies,

noncanonical NFKB activation has been observed
in pig models of ischemia-reperfusion renal injury,
possibly indicating a role for this signaling pathway
in the development of nephropathy (25). The
significance of noncanonical NFKB activation in
the hearts of our diabetic mice remains unclear and
awaits further investigations.
Our observation that TNF-a and TNFa-receptor
were increased in the hearts ofdiabetic mice indicates
chronic activation of the innate immune response
via prolonged activation of the NFKB pathway. The
possibility that infiltrating neutrophils, monocytes,
and macrophages also are responsible for cytokine
production cannot be ruled out, but it appears unlikely
given the low numbers of infiltrating macrophages or
lymphocytes in the heart tissue.
In db/db mice, lipid accumulation in ventricular
cardiac myocytes and increased depots in fat pads
around the ventricles occurs with development of
cardiac dysfunction before cardiomyopathy and
fibrosis are microscopically detectable. Similar
findings have been observed in leptin-deficient
obese fa/fa rats (26)..Although the physiological role
of lipids in the db/db heart may be simply to store
energy, excess lipid accumulation may contribute to
oxidative stress and cardiac dysfunction through direct
activation of NFKB and expression of inflammatory
cytokines. Alternatively, lipid accumulation can
affect the biomechanical function of the heart, eg, by
affecting Ca 2+ -binding to the sarcolemmal membrane
(27) or increasing chamber and myocardial stiffness.
Study ofthe end-diastolic pressure-volume and stressstrain relationship in isolated hearts from both lean
and obese rabbits and in a murine model of isolated
cardiac steatosis at different end-diastolic volumes
has shown markedly decreased diastolic compliance
due to increased ventricular-chamber and myocardial
stiffness (28,29). The stiffer ventricles per se impair
cardiac function, as higher filling pressure is required
to expand the ventricles and ensure adequate filling
and contraction. The concurrent' increase in atrial
pressures not only impairs venous return to the heart
but also increases intramyocardial capillary pressures
and cardiac wall edema, which evolves into fibrosis
over time (30).
In humans, obesity is independently associated
with an increase in morbidity and mortality, resulting

71

primarily from stroke, coronary artery disease, and

congestive heart failure (31). Interestingly, some
obese patients with symptoms of systemic and
pulmonary congestion have normal systolic function;
however, diastolic function is often abnormal (32),
and 35% to 40% of patients with congestive heart
failure may have diastolic dysfunction only (33).
Furthermore, diastolic dysfunction and pulmonary
hypertension can be worse after exercise. In dogs
given a high-fat diet for 4 weeks, the resulting
weight gain led to a significant increase in left atrial
pressure, both at rest and (more importantly) during
exercise, and to a net decrease in exercise tolerance
(34). In addition, the hearts of obese rabbits exhibit
mild systolic dysfunction, with reduced contractile
responsiveness to isoproterenol (35). In our study,
echocardiography with Doppler flow analysis
revealed pronounced changes in the cardiac diastolic
function of db/db mice after exercise: specifically,
the peak height of the A-wave increased, while
the E/A ratio decreased, reflecting impaired LV
relaxation during diastole. In the Otsuka Long
Evans Tokushima Fatty (OLETF) rat model of type
2 diabetes, diastolic cardiac dysfunction develops
in parallel with interstitial collagen accumulation
(36). In our study, however, we detected no gross
differences in interstitial collagen deposition in the
hearts of db/db mice. Furthermore, LV mass and
relative wall thickness were unchanged, suggesting
that diastolic dysfunction was not paralleled by
cardiac hypertrophy in db/db mice but was solely
imputed to lipid accumulation within and around
the heart. Fractional shortening was similar to that
observed in db/db mice at baseline. However, BNP
mRNA expression was significantly increased in db/
db versus nondiabetic mice. These data suggest that
marked lipid accumulation in db/db mouse hearts
is associated with cardiac diastolic dysfunction (of
the chamber rather than wall) without evidence of
systolic dysfunction, at least up to 12 months of age.
This finding agrees with the clinical observation
that impairment of diastolic function precedes
impairment of systolic function by decades in
humans with diabetes (37).
In conclusion, spleen inflammatory responses
are associated with expression of TNF-a receptor
type 1 and strong activation of NF-KB, significant
alteration of canonical RelA/p65, and (to a lesser

72

R. MADONNA ET AL.

extent) expression of the noncanonical RelB/p52

subunit in the hearts of diabetic mice. These hearts
also displayed a markedly increased lipid content,
which may at least partially reflect activation of
NFKB and induction of cardiac proinflammatory
genes. Echocardiography with Doppler flow analysis
and measurements of cardiac BNP expression
revealed diastolic dysfunction with no signs of
systolic dysfunction. Therefore, in obese db/db mice,
abnormal immune responses and TNF-a production
in spleen tissue, along with cardiomyocyte lipid
accumulation, may play a role in NFKB activation
in the heart, with consequent development and
progression of diastolic cardiac dysfunction.
ACKNOWLEDGEMENTS
This study was supported by funds from the
National Institutes of Health (grants ROIHL59249
and ROIHL69509 to YJG), the Texas State Higher
Education Coordinating Board ATP/TDP program
(YJG), and the United States Department of Defense
(YJG). We thank Dr. Deborah Vela for helping with
the histopathologic studies. We also thank Ms. Roxy
Tate and Virginia Catherine Fairchild for helping to
proofread and edit this manuscript.

6.

7.

8.

9.

10.
11.

12.

13.

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