J. Biomedical Science and Engineering, 2011, 4, 506-510
doi:10.4236/jbise.2011.47064 Published Online July 2011 (http://www.SciRP.org/journal/jbise/
JBiSE
).
Published Online July 2011 in SciRes. http://www.scirp.org/journal/JBiSE
Uncoupling protein 2 regulates myocardial apoptosis via the
diabetogenic action of streptozotocin
Xiu-Zhen Li1, Ruo-Yun Tan2, Xiang Lu1＊
1The First Clinical College, NJMU, Nanjing, China;
2The Second Clinical College, NJMU, Nanjing, China.
Received 10 January 2010; revised 26 April 2010; accepted 24 May 2011.
ABSTRACT
Objective: Determine the role of uncoupling protein 2
(UCP2) in the myocardial apoptosis of diabetic melli-
tus（DM）. Methods: DM animal models were induced
by streptozotocinon (STZ) on UCP2 knock-out mice
(UCP2KO) and wild-type mice (WT), which were
reared for 7 and 28 days after successful modeling,
respectively. The expressions of relative protein for
myocardial apoptosis, pro-caspase-9, were investigated
using western blot. However, the terminal deoxynu-
cleotidyltransferase-mediated dUTP-biotin nick end
labeling (TUNEL) was used to explain apoptosis at the
DNA level. Results: Image analysis showed that the
expression of pro-caspase-9 protein levels increased
slightly in UCP-/- + DM-7-day group comparing with
DM-7-day group (P > 0.05). The expression of
pro-caspase-9 protein levels increased significantly (P
< 0.05）in UC P-/- + DM-28- day group comparin g with
DM-28-day group. TUNEL analysis indicated that
UCP2 reduced the number of apoptotic myocytes in
the DM-28-day group by 70% in comparison to
DM-7-day group by 30% (P < 0.05). Conclusion UCP2
may be one of the most important factors that con-
tribute to the myocardial apoptosis of DM.
Keywords: Uncoupling Protein 2; Diabetes;
Myocardium; Apoptosis
1. INTRODUCTION
An inner mitochondrial protein known as uncoupling pro-
tein 2 (UCP2) is a member of the uncoupling protein fam-
ily and belongs to the inner mitochondrial membrane an-
ion-carrier superfamily. UCP2 can be detected in the heart
[1], brain, lung, spleen, kidney, liver, and adipose tissue.
UCP2 is a regulator of reactive oxygen species during
electron transport in the mitochondrial inner membrane,
plays an active role in the prevention of atherosclerosis [2],
is one of the etiologies of type 2 diabetes [3], participates
in inflammation [4], and regulates cell apoptosis [5]and
aging [6]. The purpose of this study was to investigate the
relationship between UCP2 and the myocardial apoptosis
of diabetic mellitus, demonstrate the important role of
UCP2 in diabetic cardiomyopathy (DC), and provide ad-
ditional theoretical foundation for DC therapy.
2. MATERIALS AND METHODS
2.1 Animals
Male, 8- to 12-week old WT and UCP2KO littermate
mice were used in this study. UCP2KO and WT mice
were generated on a C57B/6 mixed background. The
WT mice were purchased from the Shanghai Silaike
Laboratory Animal Company. UCP2KO mice were pre-
viously generated by homologous recombination in em-
bryonic stem cells on a C57BL/6 mixed background in
the Laboratory Animal Center of Nanjing University,
wherein their genomic loci were analyzed using PCR, as
previously described [7]. The WT and UCP2KO mice
were raised in a colony at the Nanjing Medical Univer-
sity, wherein all of the animal protocols were approved
by the Animal Care Committee, and the animals were
handled according to the guidelines of the Chinese
Council of Animal Care.
2.2. Chemicals and Reagents
STZ (N-(methylnitrosocarbamoyl)-α-D-glucosamine),
anti-caspase9 antibody, and a TUNEL Apoptosis Assay
Kit was obtained from Bioworld.
2.3. STZ-Induced Diabetes
Each mouse received a single daily injection of STZ (60
mg/kg) for three consecutive days. Animals in the nega-
tive control group (NC) and blank control group (BC)
were identically handled, but were injected with only
vehicle (citrate buffer). All STZ-Injected animals were
assessed for the development of diabetes as described
previously [8] and sacrificed on days 7 or 28, and their
tissues were harvested for further analyses.

X.-Zhen Li et al. / J. Biomedical Science and Engineering 4 (2011) 506-510 507
2.4. Blood Glucose Concentration
Measurements
Plasma glucagon concentrations were assayed using a
glucometer (Johnson & Johnson) according to the
manufacturer’s instructions.
2.5. Western Blotting
Heart tissues were immediately homogenized in Lysis
Buffer at 4˚C as described previously [9]. Insoluble ma-
terial was removed by centrifugation for 30 min at
14,000 rpm at 4˚C. The protein concentration of apop-
tosis protein was measured using a bicinchoninic acid
(BCA) kit and standardized for 60 μg/ml. The total pro-
tein was separated by 12% separating gel with a 3.9%
stacking gel. Next, the gel was electro transferred onto
nitrocellulose membranes (Trans-Blot, Bio-Rad) and
blocked with 5% skim milk for 2 h. The membrane was
incubated in primary antibody (anti-pro-caspase-9, 1:600)
and diluted in 0.1% Tween Tris-buffered saline (TBST)
overnight at 4˚C. Subsequently, the membrane was in-
cubated in secondary antibody (anti-rabbit IgG HRP
conjugate, 1:800) and diluted in 0.1% TBST for 1 h at
room temperature. Specific signals were detected using
enhanced chemiluminescent (ECL) plus reagent (Bio-
Rad). The protein loading was 30 μl and was normalized
using a housekeeping gene antibody (mouse anti β-actin
at 1:10000 and anti-mouse IgG HRP at 1:5000).
2.6. Apoptosis and Immunohistochemistry
Small (<1.0 cm) pieces of heart remnants were fixed
overnight at 4˚C in a solution of 4% paraformaldehyde
in phosphate-buffered saline. The tissue was dehydrated,
paraffin-embedded, sliced (4-μm thickness), dewaxed,
and hydrated. Apoptosis was assessed via the TUNEL
method using a ApopTag fluorescein in situ detection kit
(Bioworld). Labeling indices were determined without
previous knowledge of the genotype of the mice by
counting the number of TUNEL-positive cells per 40
myocytes in each field. There were at least five fields for
each slice and six slices for each animal.
3. STATISTICAL ANALYSIS
The data are expressed as mean ± S.E.M, where applica-
ble. The significance of the data was evaluated using
univariate analysis via the Tukey-Kramer test or Dunn’s
multiple comparisons post test, where applicable. Dif-
ferences were considered as being statistically signifi-
cant at P < 0.05.
4. RESULTS
4.1. The Effect of UCP2 on the Development of
Hyperglycemia in STZ-Treated Mice
WT and UCP2KO were compared to evaluate how the
absence of UCP2 affects the development of hypergly-
cemia. Blood glucose was measured at the time of suc-
cessful modeling, in order to monitor the development of
the STZ-induced hyperglycemia. Both control (normal
saline only) groups exhibited consistent blood glucose
homeostasis over the 7-day and 28-day study periods
and did not show any significant difference or change in
blood glucose concentration in comparison to their
starting values. After successful modeling, the blood
glucose levels in both the WT and UCP2KO mice were
not significantly different; however, after day 28, STZ-
treated UCP2KO mice demonstrated less hyperglycemia
development in comparison to WT mice (Figure 1).
4.2. The Effect of UCP2 on Myocardial
Apoptosis after STZ Treatment
Myocardial apoptosis was measured via Western blot
analysis of apoptotic protein and TUNEL staining on
paraffin sections.
The expression of pro-caspase-9 was observed to in-
crease in the UCP-/- + DM-7-day group in comparison
to the DM-7-day group, although this increase was not
statistically significant (P > 0.05), whereas the expres-
sion of pro-caspase-9 was observed to increase in the
UCP2-/- + DM-28-day group in comparison to the
DM-28-day group, wherein these results were statisti-
cally significant (P < 0.05, Figure 2). The number of
TdT-labeled myocytes decreased 2.0-fold, from 71.00 ±
0.087% in the UCP2KO + DM-28-day group to 30.10 ±
0.079% in the DM-28-day group (P < 0.05), whereas
decreased 1.2-fold, from 12.98 ± 0.050% in the
UCP2KO + DM-7-day group to 9.84 ± 0.079% in the
DM-7-day group (P > 0.05) (Figure 3).
5. DISCUSSION
DM is a major risk factor for the development of several
cardiovascular complications, which in turn have be-
Figure 1. Blood glucose levels in the
different groups, for A, B, C, D, E, F,
G.
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X.-Zhen Li et al. / J. Biomedical Science and Engineering 4 (2011) 506-510 508
Figure 2. The expression of pro-caspase9 at different time (7-day, 28-day) in different
groups, for A, B, C, D, E, F, G.
(a) (b) (c) (d)
(e) (f) (g) (h)
Figure 3. The result of TUNEL (10 × 40), respectively, A: NC7-day；B: DM7-day; C：U-/-7-day; D:
UCP2-/- + DM7-day E:NC28-day; F: DM28-day; G: U-/-28-day; H: UCP2-/- + DM28-day; *P < 0.05
versus wild-type.
come the primary causes of death in the diabetic popula-
tion [10]. Hyperglycemia is considered to be the primary
cause of most chronic diabetic complications, such as
cardiovascular pathological changes, diabetic retinopa-
thy, diabetic nephropathy, diabetic foot, etc., which oc-
curs via the deregulation of β-cell insulin secretion and
the development of peripheral tissue insulin resistance.
DC was first reported in 1972 by Rubler et al. [11],
who reported the autopsy data obtained from four pa-
tients with diabetic renal microangiopathy and dilated
left ventricles in the absence of other common causes.
Therein, they observed that there was a close relation-
ship between myocardial apoptosis and the occurrence
or development of diabetic cardiomyopathy. In the mi-
tochondrial-initiated pathway, mitochondrial cytochrome
c release and the activation of caspase play important
roles in diabetes-induced cardiac cell death. Caspase
activation is triggered by the formation of a multimeric
Apaf-1/cytochrome c complex that is fully functional in
the recruitment and activation of pro-caspase-9, resulting
in further apoptosis.
In order to further investigate the role of UCP2 in the
development of diabetes, we treated both WT and
UCP2KO mice with STZ injections as to experimentally
induce diabetes. We observed that the UCP2KO mice
developed hyperglycemia, although significantly less
severe in comparison to the WT group.
After four weeks, the myocardial mitochondria of DM
mouse began to swell and denature [12]; On days 7, 14,
and 21 after a single dose of STZ (150 mg/kg) treatment,
the number of TUNEL-positive cells and the amount of
caspase-3 activation increased [13]; Twenty-eight days
after successful modeling (STZ treatment, 60 mg/kg),
cardiac apoptosis in the Sprague Dawley（SD) rats were
observed to have increased (P < 0.01) [14] ; The number
of cardiac myocytes,which cultured by different blood
glucose level, associated with blood glucose concentra-
tion; and Hyperglycemia through mitochondrial pathway
resulted in myocardial apoptosis [15]. Our previous
work has indicated that, even though the expression of
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X.-Zhen Li et al. / J. Biomedical Science and Engineering 4 (2011) 506-510 509
cleaved-caspase-3 increased, in addition to cell shrink-
age and nuclear condensation in the DM-7-day group in
comparison to the WT group , this increase was not sta-
tistically significant (P > 0.05), which is consistent with
our reported experimental data.
UCP2 plays a significant role in the onset of diabetes,
specifically via the regulation of cellular ATP produc-
tion [16], islet beta-cell apoptosis [17] and reduction
ROS production [18]. UCP2 also affects the occurrence
or development of DM and participates in the myocar-
dial apoptosis of DC. UCP2KO developed lesser hyper-
glycemia than their WT counterparts after multiple
low-dose STZ injections in vivo [19]. TUNEL analysis
indicated that the number of apoptotic myocytes in the
UCP2KO + DM28-day group decreased by 30% in com-
parison to in the DM-28-day (P <0.05), while the num-
ber of apoptotic myocytes in the UCP2KO + DM7-day
group decreased by 0.2% in comparison to the
DM-7-day (P > 0.05). Our previous work has indicated
that cleaved-caspase-3 expression markedly decreases in
UCP2KO + DM-28-day mice，wherein chromatin was
condensed with high intensity fluorescence in compari-
son to DM-28-day mice, with a statistical significance (P
< 0.05); however, the expression of the cleaved-cas-
pase-3 slightly decreased in the UCP2-/- + DM-7-day
group, wherein chromatin was condensed with low in-
tensity fluorescence, in comparison to the DM-7-day
group, although the results were not statistically signifi-
cant (P > 0.05). Therefore, it can be concluded that
UCP2 reduces the number of apoptotic myocytes and
can provide time-dependent anti-apoptosis activity via
variable caspase-3 or caspase-9 expression.
In this article, myocardial apoptosis slightly increased
in the negative control group in comparison to the blank
group, although this increase was not statistically sig-
nificant (P > 0.05). Although cardiac mitochondrial
UCP2-mediated uncoupling respiration was impaired,
short-term mitochondrial efficacy was increased, which
(7-day or 28-day) improved the function of the myocar-
dium.
Diabetic cardiomyopathy results in myocardial dys-
function. Meanwhile，it has been reported that the loss of
myocardial cell quality and quantity could act as an im-
portant factor in heart dysfunction because they are
non-renewable. UCP2 plays a crucial role in mitochon-
drial energy metabolism and in the occurrence or devel-
opment of DC. Furthermore, it is reported that UCP2 is
involved in determining lifespan [20]. Further research
into UCP2 should investigate the relationship between
UCP2 and myocardial apoptosis, and therein, attempt to
identify the causes so as to improve treatment protocols
and provide a more developed basis for the diagnosis
and treatment of DC.
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