# Evaluation of Bcl-2 Expression and Caspase-3 in  STZ-Induced Diabetic Rats

Authors: Iraj Jafari Anarkooli; Mojtaba Sankian; Shahriar Ahmadpour; Abdol-ReZa Varasteh; Hossein Haghir
Journal: Experimental Diabetes Research
 (2008)

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## Abstract

We assessed the expression of Bcl-2 family members at both mRNA and protein levels as well as the Caspase-3 activity, in order to investigate the occurrence of apoptosis in hippocampus of STZ-induced diabetic rats. We selected twenty-four Wistar rats; half of them were made diabetic by intraperitoneal injection of a single 60 mg/kg dose of streptozotocin (STZ, IP), while the others received normal saline and served as controls. The expressions of Bcl-2,Bcl-xL, and Bax mRNA and proteins were measured using RT-PCR and western blotting, respectively. Caspases-3 activity was determined by using the Caspase-3/CPP32 Fluorometric Assay Kit. The result showed that mRNA
and protein levels of Bcl-2 and Bcl-xL were lower in hippocampus of diabetic group than that of the control group, whereas expressions of Bax in hippocampus of diabetic rats were higher than that of controls at both mRNA
and protein levels (P<.01). Hyperglycemia was found to raise 6.9-fold hippocampal caspase-3 activity in diabetic group compared with control group (P<.001). Therefore, the induction of diabetes is associated with increased ratios of Bax/Bcl-2, Bax/Bcl-xL, and increased caspase-3 activity in hippocampus which shows that apoptosis is favored in hippocampal region.


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## Body

## 1. Introduction

Diabetes  mellitus is one of the most severe problems global,
which is rising significantly. At the  present, the WHO accounts 177 million
patients  with diabetes, while this number is expected to double in 2030 [1]. Diabetes
mellitus is a heterogeneous metabolic disorder diagnosed by hyperglycemia
resulting from impaired insulin secretion, resistance to insulin action, or
both [2]. Hyperglycemia, which occurs under the diabetic condition, is often
associated with complications, such as cardiovascular disease, renal failure,
retinopathy and peripheral, and autonomic neuropathy [3, 4]. In recent years, it
has been suggested that diabetes also affects the central nervous system [5, 6].
Within the central nervous system, the hippocampus is considered as a particular
target for changes related to diabetes [7]. Hippocampus is a part of
limbic system that serves as a critical integration center for cognitive
functions such as learning and memory in the mammalian brain [8]. It has been reported that impairments in
memory, learning, and cognition are more common in diabetic people than in
nondiabetic ones. In type-1 diabetes mellitus, hippocampal neurons are
extremely vulnerable [9–14]. Little is known about
the mechanism by which hyperglycemia affects neurons [15].Apoptosis
could be proposed as a possible mechanism for hyperglycemia-induced hippocampal
neuronal cell death. It has  been revealed that is apoptosis  implicated  in several neurodegenerative disorders
like Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, and
amyotrophic lateral sclerosis [11, 16]. Apoptosis is a gene-regulated occurrence
characterized by special morphological features, including condensation of
chromatin, shrinkage of cell and nucleus, membrane blebbing, and DNA
fragmentation [17]. Several factors are contributed in apoptosis, but the key
elements are categorized in two main families of proteins including caspase
enzymes and Bcl-2 family [18]. Caspase enzymes are working as a cascade and
caspase 3 is the most important member of this family which plays an effective
role in apoptosis of neurons of central nervous system [19]. Bcl-2 family is a
set of cytoplasmic proteins that regulate apoptosis. The two main groups of
this family, Bcl-2 and Bax proteins, are functionally opposed: Bcl-2 and Bcl-xL act to inhibit apoptosis, whereas Bax counteracts this effect [19, 20].In this study, we therefore assessed
the expression of Bcl-2 family members at both mRNA and protein levels as well
as the Caspase-3 activity, in order to investigate the occurrence of apoptosis
in hippocampus of STZ-induced diabetic rats.


## 2. Materials and Methods

We
selected twenty-four male Wistar rats weighing (200–250 g) for this study.
Animals were randomly divided into two groups (n = 12 in each group): the control
(C) and diabetic (D) groups. They were kept in the animal house (Avicenna Research
institute, Mashhad University of Medical Sciences, Mashhad, Iran) for one week for proper acclimatization before starting
the experiment under controlled condition of illumination (12 hours light/12 hours
darkness) and temperature 23 ±
2°C. They were
housed under ideal laboratory conditions, maintained on standard pellet diet
and water ad libitum throughout the experimental period. All procedures
were in accordance with the Guide for the Care and Use of Laboratory Animals of
Mashhad University of Medical Sciences, Iran.Diabetes was induced in overnight fasted experimental group by a single intraperitoneal injection of
STZ (Sigma, 60 mg/kg body weight) dissolved in normal saline, while the control
group was injected
with the normal saline only [21]. Three days after administration of STZ, the
tail vein blood glucose level was measured in all animals. Blood glucose levels
of 250 mg/dl and above were considered diabetic [22]. At the end of the eighth
week, we measured weight and blood glucose level of rats. Then, animals were
anesthetized with chloroform and the skull was open along the midline and the
brain was removed and placed on an ice-cooled cutting board. The meninges were
carefully removed and hippocampus was dissected from hemispheres, snap frozen
in liquid nitrogen and stored at −70°C for extraction of DNA, RNA, and protein.


### 2.1. Isolation of Total RNA and Synthesis of cDNA

RNA was isolated from processed hippocampal tissue of rats using TriPure Isolation
Reagent (Roche, Germany) according to the
manufacturer instructions. Total RNA was
reverse transcribed (RT) into cDNA with MBI RevertAid (Fermentase, Life
Sciences, Lithuanian) according to the manufacturers instructions, and cDNA samples were stored
at –20°C.


### 2.2. RT-PCR

A semiquantitative reverse transcriptase polymerase
chain reaction (RT-PCR) was carried out to determine
the levels of Bcl-2, Bcl-xL, and Bax mRNA expressions. The
RT-PCR mixture (final volume of 20 μL) contained 3 μL of cDNA, 10 μL of Qiagen
Multiplex PCR Master Mix 2x (Qiagen, Germany) and 10 pmols of each
complementary primer specific for Bcl-2, Bcl-xL, and Bax sequences as
well as for GAPDH (Glyceraldehydes-3-phosphate
dehydrogenase; GAPDH gene) sequence as an internal control (Table 1). The
samples were denatured at 95°C for 15 minutes, and amplified using 40 cycles
of 95°C for 30 seconds, 60°C for 80 seconds, and 72°C for 45 seconds for Bax
gene and 5 cycles of 95°C
for 30 seconds, 62°C
for 60 seconds, and 72°C
for 30 seconds, as well as 25 cycles of 95°C for 30 seconds, 64°C for 80 seconds and 72°C for 30 seconds for Bcl-2 and
Bcl-xL genes followed by a final elongation at 72°C for 3 minutes
on a Corbett Research thermocycler (Sydney, Australia). The optimal numbers of cycles have been selected for amplification of all three genes experimentally so that amplifications were in the exponential range and have not reached a plateau. Five microliters of the
final amplification product were run on a 3% ethidium-stained agarose gel and
photographed. Quantification of the results
was accomplished by measuring the optical density of the labeled bands, using
the computerized imaging program Kodack-1D (USA). The values were normalized to
GAPDH intensity levels.Table 1
Primers used for RT-PCR method for Bcl-2, Bcl-xL, Bax, and GAPDH.
GenesPrimersProduct sizeBcl-2F:5′-CTG GTG GAC AAC ATC GCT CTG-3′228 bpR:5′-GGT CTG CTG ACC
TCA CTT GTG-3′Bcl-xLF:5′-AGG CTG GCGATG AGT TTG AA-3′357 bpR:5′-TGA AAC GCT CCT GGC CTT TC-3′BaxF:5′-TTCATC CAGGAT CGA GCA GA-3′263 bpR:5′-GCA AAG TAG
AAG GCA ACG-3′GAPDHF:5′-GGCCAAGAT CAT CCA TGA CAA CT-3′462 bpR:5′-ACC AGG ACA TGA GCT TGA CAA AGT-3′


## 2.1. Isolation of Total RNA and Synthesis of cDNA

RNA was isolated from processed hippocampal tissue of rats using TriPure Isolation
Reagent (Roche, Germany) according to the
manufacturer instructions. Total RNA was
reverse transcribed (RT) into cDNA with MBI RevertAid (Fermentase, Life
Sciences, Lithuanian) according to the manufacturers instructions, and cDNA samples were stored
at –20°C.


## 2.2. RT-PCR

A semiquantitative reverse transcriptase polymerase
chain reaction (RT-PCR) was carried out to determine
the levels of Bcl-2, Bcl-xL, and Bax mRNA expressions. The
RT-PCR mixture (final volume of 20 μL) contained 3 μL of cDNA, 10 μL of Qiagen
Multiplex PCR Master Mix 2x (Qiagen, Germany) and 10 pmols of each
complementary primer specific for Bcl-2, Bcl-xL, and Bax sequences as
well as for GAPDH (Glyceraldehydes-3-phosphate
dehydrogenase; GAPDH gene) sequence as an internal control (Table 1). The
samples were denatured at 95°C for 15 minutes, and amplified using 40 cycles
of 95°C for 30 seconds, 60°C for 80 seconds, and 72°C for 45 seconds for Bax
gene and 5 cycles of 95°C
for 30 seconds, 62°C
for 60 seconds, and 72°C
for 30 seconds, as well as 25 cycles of 95°C for 30 seconds, 64°C for 80 seconds and 72°C for 30 seconds for Bcl-2 and
Bcl-xL genes followed by a final elongation at 72°C for 3 minutes
on a Corbett Research thermocycler (Sydney, Australia). The optimal numbers of cycles have been selected for amplification of all three genes experimentally so that amplifications were in the exponential range and have not reached a plateau. Five microliters of the
final amplification product were run on a 3% ethidium-stained agarose gel and
photographed. Quantification of the results
was accomplished by measuring the optical density of the labeled bands, using
the computerized imaging program Kodack-1D (USA). The values were normalized to
GAPDH intensity levels.Table 1
Primers used for RT-PCR method for Bcl-2, Bcl-xL, Bax, and GAPDH.
GenesPrimersProduct sizeBcl-2F:5′-CTG GTG GAC AAC ATC GCT CTG-3′228 bpR:5′-GGT CTG CTG ACC
TCA CTT GTG-3′Bcl-xLF:5′-AGG CTG GCGATG AGT TTG AA-3′357 bpR:5′-TGA AAC GCT CCT GGC CTT TC-3′BaxF:5′-TTCATC CAGGAT CGA GCA GA-3′263 bpR:5′-GCA AAG TAG
AAG GCA ACG-3′GAPDHF:5′-GGCCAAGAT CAT CCA TGA CAA CT-3′462 bpR:5′-ACC AGG ACA TGA GCT TGA CAA AGT-3′


## 3. Western Blot Analysis

Tissues collected were
homogenized with ice-cold homogenizing buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM
EDTA, and 0.5 mM Triton X-100, PH 7.4) and protease inhibitor cocktail tablets (Roche,
Germany). Proteins were measured with Bio-Rad protein assay method. Hippocampal
protein lysates (50μg/well) were separated by SDS-PADE (12.5%)
under reducing conditions and transferred to a polyvinylidene difluoride (PVDF)
membrane (Millipore, USA).
Membranes were treated with Attoglow western blot system kit, according to the
manufacturer protocol (Biochain,
USA). Briefly,
blots were blocked with blocking buffer (5% not-fat dried milk in PBS). After
blocking, blots were incubated with anti-Bcl-2 polyclonal antibody (1/500,
v/v), anti-Bcl-xL polyclonal antibody (1/1000, v/v), and anti-Bax
polyclonal antibody (1/250, v/v) (Biovision, USA) for 20 hours at 4°C. Blots were washed for 4 times with 0.1% tween 20 in PBS
and incubated with HRP conjugated-secondary antibody (1/5000, v/v, Biochain,
USA) for 1 hour at room temperature. The Bcl-2, Bcl-xL and Bax
protein bands were visualized using enhanced chemiluminescnces (ECL) method
(Bioimaging, system, syngene, UK).


## 4. Detection of Caspase-3 Activity

Caspase-3 activity was
determined using the Caspase-3/CPP32 Fluorometric Assay Kit (K105) purchased
from Biovision, Inc. (Mountain View,
Calif., USA).
For each assay, 50μg of tissue cell lysate was used. Samples were read in a
fluorimeter equipped (Jasco, FP-6200, Japan) witha 400-nm
excitation and a 505-nm emission filter. Fold-increasein Caspase-3
activity was determined by comparing fluorescence of 7-amino-4-trifluoromethyl coumarin in control and treated hippocampus with STZ.


## 5. Statistical Analyses

All
results are evaluated
using themean±S.E.M. The results were analyzed with Mann-Whitney and paired t-tests. A probability level of P <.05 was
considered significant.


## 6. Results

Blood glucose concentrations
of diabetic rats were more than 250 mg/dl 3 days after the administration of
STZ. Blood glucose level at the end of the 8th week was significantly increased
in diabetic group compared to nondiabetic (control) group (P<.001).At first, both groups had similar body
weights. Body weight was increased in the control group at the end of 8 weeks;
however, the diabetic groups lost body weight compared to the control ones (P<.01) (Table 2).Table 2
Characteristic
parameters of the control and STZ-diabetic rats. Values  are shown asmean±S.E.M. of 12 rats per group.
ControlDiabeticInitial body weight (g)243.7±9.2247.0±19.0Final body weight (g)306.0±10.4198.1±21.0*Initial blood glucose
(mg/dl)109.4±5.6106.9±6.3Final blood glucose
(mg/dl)111.2±1.3477.1±28.4***P<.01
versus control.**P<.001
versus control.


## 6.1. Expression of Bax, Bcl-2 and Bcl-xL  Genes at mRNA Level

As shown in Figure1, expression
of Bax was increased in the hippocampus of STZ-induced diabetic group compared
to that of control group, while the expressions of
Bcl-2 and Bcl-xL were reduced. Furthermore, the Bax/Bcl-2 and the
Bax/Bcl-xL ratios were increased in the hippocampus of STZ-induced
diabetic group compared to control group (Figures 2(a) and 2(b)).Semiquantative multiplex RT-PCR analysis of expressions of Bax,
Bcl-2, and Bcl-xL genes in hippocampus of control (C) and
diabetic (D) groups of rat. (a)
Amplification of the Bax gene (263 bp) compared with GAPDH gene (461 bp). (b)
Amplification of the Bcl-2 gene (228 bp) compared with GAPDH (461 bp). (c)
Amplification of the Bcl-xL gene (357 bp) compared with GAPDH
(461 bp).
(a)(b)(c)(a)  Bax/Bcl-xL ratio of control (C) and STZ-induced diabetic (D)
groups of rat. Bar graph indicates the mean±S.E.M.#P<.01, compared to the control group (n = 4 each
group).  (b)  Bax/Bcl-2 ratio of control (C) and STZ-induced diabetic
(D) groups. Bar graph indicates the mean±S.E.M.#P<.05, compared to control group  (n = 4 each group).
(a)(b)


## 6.2. Expressions of Bax, Bcl-2, and Bcl-xL Proteins

After eight weeks, the expression
of proapoptotic Bax protein was enhanced in hippocampus of STZ-induced diabetic
group compared to control group of rats. On the other hand, the expression of
anti-apoptotic Bcl-2 and Bcl-xL proteins were decreased in
hippocampus of STZ-induced diabetic group compared to the control group of rats
(Figure 3).Figure 3
Western blot analysis to determine the expression of Bax, Bcl-2 and
Bcl-xL in extracts from hippocampus of control (C) and diabetic
(D) groups of rat.


## 6.3. Evaluation of Caspase-3 Activity in Tissue Extracts

Caspase-3 activity was
significantly higher in STZ-induced diabetic group compared to that of nondiabetic
group (174.2 ± 36 versus  25 ± 10.25,P<.001). Hyperglycemia was found to increase
6.9-fold hippocampal tissue caspase-3 activity in diabetic group compared with
nondiabetic group (Figure 4).Figure 4
Caspase-3 activity in hippocampal tissues of control (C) and
STZ-induced diabetic (D) rat groups. Bar graph indicates themean±S.E.M.#P<.001, compared to the control group (n = 4 each
group). Fold-increasein Caspase-3
activity was determined by comparing fluorescence of
7-amino-4-trifluoromethyl courmarin in control and treated
hippocampus with STZ.


## 7. Discussion

Despite of extensive investigations, the mechanism(s) activating
apoptotic pathways in the diabetic brain have not been completely understood [23].
In the present study, we showed the effects of hyperglycemia on the expression of Bcl-2 family protein levels and Caspase-3
activity in hippocampus
of STZ-induced diabetic rats.Our  results  showed that after 8 weeks, Bax expression was considerably
increased in hippocampus of STZ-induced diabetic rats at both mRNA and protein
levels, while the expressions of Bcl-2 and Bcl-xL were significantly
reduced at both mRNA and protein levels. Furthermore, Bax/Bcl-2 and Bax/Bcl-xL ratios, a main index of apoptotic cell death, were significantly increased; signifying
hyperglycemia-induced apoptosis in hippocampus of STZ-induced diabetic rats
could possibly be mediated by the mitochondrial pathway [24].Other studies have
demonstrated that diabetes induces apoptosis in central nervous system as well
as in neuronal-related tissues, including peripheral nervous system, ganglions,
and retina [11, 25, 26]. Several
experimental models support that overexpression of Bax can induce apoptosis in
cells both in vivo and in vitro [11, 27]. In addition, the apoptosis has been
reported in neuroblast cell cultures treated with diabetic human sera [28].
Sharifi et al. showed that the excess concentration of glucose resulted in an increase
in Bax expression protein and then induced apoptosis in PC12 cells [15].
Apoptosis and decrease in Bcl-2 expression were detected in primary neurons of dorsal root ganglion of spinal cord in
diabetic rats [29, 30].In this study, we were able to reveal the activation of proapoptotic
proteins and suppression of antiapoptotic proteins in hippocampus of
STZ-induced diabetic rats at both mRNA and protein levels. There are two
classes of regulatory proteins in Bcl-2 family that substantiate opposite effects
on apoptosis: the antiapoptotic members including Bcl-2 and Bcl-xL which
protect cells against some forms of apoptosis, and proapoptotic members
including Bax, bak, and Bcl-xS which progress programmed cell death.
Apoptotic signals converge toward a common death pathway, for which caspases perform
apoptosis and the Bcl-2 family proteins regulate it [31]. There is an absolute evidence that three main signals
cause the release of apoptogenic mitochondrial mediators including a
proapoptotic member of the Bcl-2 family, elevated level of intracellular
calcium, and reactive oxygen species (ROS) or free radicals [32]. It has previously
been revealed that both ROS and Nitric Oxide (NO) can change mitochondrial
membrane potential by opening the mitochondrial permeability transition pores
(mPTP), releasing cytochrome C and subsequent activation of caspases 9 and 3
and finally causing cell death. Members of the Bcl-2 family are known to be pro-
and anti-apoptotic. The balance between pro- and anti-apoptotic signals from
this family has a central role in the release of cytochrome C and its succeeding
consequences [33].Furthermore, we found that the activity of caspases-3, an effector of apoptosis that plays a key role to link the
commitment and degradation phases
[34], was significantly
increased in hippocampus of STZ-induced diabetic rats compared to nondiabetic
rats, which is consistent with previous studies [35–38].Concurrent with our findings, Vincent et al. showed
that hyperglycemia observed in diabetes mellitus was associated with oxidative
stress-induced neuronal and Schwann cells
death via increased caspase 3 activity [39].The precise mechanism through
which this occurs in hyperglycemia-induced apoptosis
is not clear, but it has shown that hyperglycemia could elevate ROS in hippocampal
neuronal cells resulted in elevation of
intracellular calcium in neuronal cells [40]. Russell et al. showed excess
mitochondrial ROS due to hyperglycemia depolarize mitochondrial membranes followed by decrease in
ATP activity and increase in Caspases-3 and 9 leading to Caspase-dependent
apoptosis [29].
Therefore, the induction of diabetes is associated with increased ratios
of Bax/Bcl-2 and Bax/Bcl-xL as well as increase of caspase-3
activity in hippocampus which shows that apoptosis is favored in
hippocampal region. In our experiment we also alternative methods such as, silver staining for dark neurons, stereology, electron microscopy and DNA laddering to support the results that obtained with Bcl-2 family gene expression and caspase-3 activity (Unpublished).


## 8. Conclusion

Taking together, it may finally be concluded that the
STZ-induced hyperglycemia causes hippocampal neuronal cell death, in which
apoptosis plays an important role possibly by an increment in the Bax/Bcl-2 and
Bax/Bcl-xL ratio, as well as increasing caspase-3 activity.
Diabetic-induced memory and cognition deficits may be partly due to a
facilitation of apoptosis and this study provide further knowledge to
mechanisms involved in STZ-induced apoptosis in hippocampus.



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*Source: 638467-2008-10-12.xml*