Int. J. Mol. Sci. 2012, 13, 16241-16254; doi:10.3390/ijms131216241
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International Journal of
Molecular Sciences
ISSN 1422-0067
www.mdpi.com/journal/ijms
Article
Alterations in Glutathione Redox Metabolism, Oxidative Stress,
and Mitochondrial Function in the Left Ventricle of Elderly
Zucker Diabetic Fatty Rat Heart
Haider Raza 1,*, Annie John 1 and Frank C. Howarth 2
1
2
Department of Biochemistry, College of Medicine and Health Sciences, United Arab Emirates
University, Al Ain, P.O. Box 17666, UAE; E-Mail:
[email protected]
Department of Physiology, College of Medicine and Health Sciences, United Arab Emirates
University, Al Ain, P.O. Box 17666, UAE; E-Mail:
[email protected]
* Author to whom correspondence should be addressed; E-Mail:
[email protected];
Tel.: +971-3-7137506; Fax: +971-3-7672033.
Received: 20 September 2012; in revised form: 31 October 2012 / Accepted: 19 November 2012 /
Published: 30 November 2012
Abstract: The Zucker diabetic fatty (ZDF) rat is a genetic model in which the homozygous
(FA/FA) male animals develop obesity and type 2 diabetes. Morbidity and mortality from
cardiovascular complications, due to increased oxidative stress and inflammatory signals,
are the hallmarks of type 2 diabetes. The precise molecular mechanism of contractile
dysfunction and disease progression remains to be clarified. Therefore, we have
investigated molecular and metabolic targets in male ZDF (30–34 weeks old) rat heart
compared to age matched Zucker lean (ZL) controls. Hyperglycemia was confirmed by a
4-fold elevation in non-fasting blood glucose (478.43 ± 29.22 mg/dL in ZDF vs.
108.22 ± 2.52 mg/dL in ZL rats). An increase in reactive oxygen species production, lipid
peroxidation and oxidative protein carbonylation was observed in ZDF rats. A significant
increase in CYP4502E1 activity accompanied by increased protein expression was also
observed in diabetic rat heart. Increased expression of other oxidative stress marker
proteins, HO-1 and iNOS was also observed. GSH concentration and activities of
GSH-dependent enzymes, glutathione S-transferase and GSH reductase, were, however,
significantly increased in ZDF heart tissue suggesting a compensatory defense mechanism.
The activities of mitochondrial respiratory enzymes, Complex I and Complex IV were
significantly reduced in the heart ventricle of ZDF rats in comparison to ZL rats. Western
blot analysis has also suggested a decreased expression of IκB-α and phosphorylated-JNK
in diabetic heart tissue. Our results have suggested that mitochondrial dysfunction and
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increased oxidative stress in ZDF rats might be associated, at least in part, with altered
NF-κB/JNK dependent redox cell signaling. These results might have implications in the
elucidation of the mechanism of disease progression and designing strategies for
diabetes prevention.
Keywords: Zucker rats; diabetes; obesity; oxidative stress; cardiomyocytes; mitochondria
Abbreviations: CYP4502E1, cytochrome P450 2E1; DCFDA, 2',7'-dichlorofluorescein diacetate;
DMNA, dimethylnitrosamine; GSH, glutathione; GST, glutathione S-transferase; GSH-Px, glutathione
peroxidase; GSSG-reductase, glutathione reductase; Cyt c ox, cytochrome c oxidase; LPO, lipid
peroxidation; ROS, reactive oxygen species; SDS-PAGE, sodium dodecylsulphate polyacrylamide gel
electrophoresis; ZDF, Zucker diabetic fatty; ZL, Zucker lean.
1. Introduction
Cardiomyopathy and other cardiovascular complications associated with increased inflammatory
and oxidative stress responses are the major causes of accelerated atherosclerosis, obesity and
diabetes [1–3]. Persistent hyperglycemia and hyperlipidemia are believed to be the main causes of
increased oxidative stress, mitochondrial dysfunctions, fibrosis and apoptosis of cardiomyocytes in
diabesity and associated complications [4–6].
The male Zucker diabetic fatty (ZDF) rat is a genetic model in which homozygous (FA/FA) animals
spontaneously develop type 2 diabetes and obesity, whereas female rats become diabetic only after
feeding high fat diet [7]. The main reason for this resistance in female Zucker rats appears to be the
presence of high concentration of antioxidant glutathione (GSH) and low oxidative stress [8]. ZDF rats
also exhibit increased insulin resistance, oxidative stress, hyperlipidemia, increased inflammatory
responses and abnormal energy metabolism which are the key features of diabesity. There are studies
which suggest that the attenuation of oxidative stress by treatment with antioxidant therapeutic drugs
or dietary products have normalized the glycemic index and metabolic complications in
diabesity [9,10]. Our previous studies, using both type 1 and type 2 diabetic models, have also
suggested that myocardial Ca2+ signaling genes and proteins have also been drastically affected in
diabetes and obesity related complications [11–14]. However, in these studies, it is not clear if these
changes are in response to increased oxidative stress due to increased reactive oxygen species (ROS)
and/or decreased GSH antioxidant metabolism. Our previous studies, using in vivo type 1 diabetes
models have strongly suggested increased oxidative stress, mitochondrial dysfunction and
compromised energy and GSH metabolism in chronic diabetic complications [15–18]. Our present aim
is, therefore, to investigate the role of oxidative stress, GSH-dependent antioxidant metabolism and
mitochondrial functions in isolated cardiac myocytes from left ventricle of ZDF rats and to compare
them with age matched Zucker lean (ZL) rats. Our results have provided a better insight of the etiology
and pathology of diabetes and obesity associated complications and have implications in designing
therapeutic approaches.
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2. Results and Discussion
2.1. Alterations in Oxidative Stress in Zucker Diabetic Rat Hearts
An increase in oxidative stress in diabetic heart myocytes was observed as shown in Figure 1A–D.
An increase in ROS production (Figure 1A) was accompanied by a moderate but significant increase in
LPO (Figure 1B) and an increase in oxidative protein carbonylation as observed by DNPH coupling of
oxidized proteins (Figure 1C). Catalase activity was found to be significantly activated (Figure 1D).
These results suggest that catalase appears to be the main enzyme involved in H2O2 clearance as we
observed no marked alteration in GSH-Px activity in ZDF rats.
Figure 1. Reactive oxygen species (ROS), lipid peroxidation (LPO), protein carbonylation
and catalase activity in Zucker diabetic fatty (ZDF) rat heart. ROS (A) was measured
fluorimetrically as described in Experimental section. NADPH-dependent total LPO (B),
DNPH-coupled protein peroxidation (C) and catalase (D) were measured as described [15–19].
Results are expressed as mean ± S.E.M. from three independent experiments and asterisks
indicate significant difference (* p < 0.05 and ** p < 0.01).
2.2. Alterations in GSH Metabolism in Zucker Diabetic Rat Hearts
As shown in Figure 2, GSH concentration was markedly (~2-fold) increased (Figure 2A) in ZDF rat
myocytes compared to ZL rat heart. Similarly, a profound increase in GSH-CDNB conjugating activity
by GST enzymes was also observed (Figure 2B). On the other hand, there was no significant increase
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in GSH-Px activity (Figure 2D) while GSSG-reductase activity was found to be significantly increased
(Figure 2C). These results suggest that GSH-dependent antioxidant defense mechanisms have been
activated in ZDF rat hearts. An increase in GSH concentration and GSH-dependent conjugation of
ROS in ZDF rat heart and increased regeneration of reduced GSH by GSSG-reductase might be
involved in protecting the cardiac myocytes from oxidative damage.
Figure 2. Glutathione (GSH) concentration and GSH metabolism in ZDF rat heart: Total
free GSH (A) and GSH metabolizing enzymes; glutathione S-transferase (GST) (B),
glutathione-reductase (GSSG-reductase) (C) and glutathione peroxidase (GSH-Px) (D)
were measured in total cardiac myocyte homogenate from left ventricles as described [15,16].
Results are expressed as mean ± S.E.M. from three independent experiments and asterisks
indicate significant difference (* p < 0.05 and ** p < 0.01).
A
C
B
D
2.3. Induction of CYP450 Activity in Zucker Diabetic Rat Hearts
Cytochrome P450 2E1 (CYP 2E1) enzyme activity was also increased significantly in ZDF rat heart
when compared to ZL rat heart myocytes (Figure 3). CYP 2E1 isoenzyme has been reported to be
involved in oxidative stress and an increase in enzyme activity has also been reported in diabetes and
obesity [16,20,21].
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Figure 3. Cytochrome P450 2E1 (CYP 2E1) activity in ZDF rat heart: CYP 2E1 activity in
ZDF and Zucker lean (ZL) rat heart left ventricle fraction was measured using
dimethylnitrosamine (DMNA) as a substrate as described before [16]. Results are
expressed as mean ± S.E.M. from three independent experiments and asterisk (*) indicates
significant difference (p < 0.05).
2.4. Alterations in Mitochondrial Respiratory Functions in ZDF Rat Hearts
A significant decrease in the activities of mitochondrial inner membrane respiratory complexes has
been observed in ZDF rat hearts (Figure 4). NADH-dependent ubiquinone oxidoreductase (Complex I)
activity was markedly reduced (42%) in ZDF rat heart myocytes when compared to ZL rat hearts
(Figure 4A). Similarly, a significant decrease in activity of the terminal respiratory enzyme,
cytochrome c oxidase (Complex IV), was also observed in ZDF rat hearts (Figure 4B). These results
have clearly suggested that mitochondrial bioenergetics (ATP production) is affected in the cardiac
myocytes of ZDF rats.
2.5. Alteration in the Expression of Oxidative Stress Marker and Transcription Regulatory Proteins in
ZDF Rat Hearts
As shown in Figure 5, SDS-PAGE and Western blot analyses have demonstrated increased
expression of oxidative stress marker proteins, HO-1, iNOS and CYP 2E1 (Figure 5A). On the other
hand the expression of cytochrome c oxidase (Cyt c ox) enzyme subunit 1 was found to be markedly
reduced suggesting inhibition of mitochondrial energy metabolism under increased oxidative stress
conditions as seen in ZDF rats. We also observed a reduced expression of phosphorylated JNK
(p-JNK) and IκB-α while no apparent change in the expression of JNK (non-phosphorylated) was
noticed (Figure 5B). These results suggest alteration in cell signaling/transcription regulation in ZDF
rat heart under oxidative stress conditions when compared to ZL rat hearts.
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Figure 4. Mitochondrial respiratory Complex I and IV activities in ZDF rat heart:
Mitochondrial respiratory enzymes complexes, Complex I (A) and Complex IV (B) were
measured in freshly prepared ventricular fractions using appropriate substrates as described in
the Experimental section. Results are expressed as mean ± S.E.M. from three independent
experiments and asterisks indicate significant difference (* p < 0.05 and ** p < 0.01).
A
B
Figure 5. Expression of oxidative stress and cell signaling marker enzymes/proteins in
ZDF rat heart. Protein (50 μg) from ZDF and ZL rat hearts were subjected to 12% sodium
dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) [22] and Western blot
analyses [23] to visualize immunoreactivity of specific marker proteins for oxidative stress
(A: HO-1, iNOS, CYP2E1 and Cyt c ox) and cell signaling (B: JNK, p-JNK and IκB-α).
β-actin was used as a loading control. R.I values indicate relative intensity (of the protein
band) using expression of the proteins in ZL as 1.0. The figures are representative of
2–3 experiments. Molecular weights shown are in kDa.
A
B
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2.6. Discussion
Our previous studies in younger (9–13 weeks) animals, the ZDF rats weighed significantly more
than controls [13]. In the present study, experiments were performed in male animals when they were
30–34 weeks of age. Bodyweight in the ZDF rats was not significantly different from controls.
Diabetes mellitus was characterized by a 4-fold increase in blood glucose. Heart function is
compromised in the ZDF rats. With age, the severity of diabetes and its complications worsens and
ZDF rats are likely to become more reliant on the use of lipids and lipid reserves to meet metabolic
requirements. This may partly account for reduced weight in aged ZDF rats. Recently, using the same
cohort of elderly Zucker diabetic rats, it was demonstrated that ventricular myocyte function was well
preserved in ZDF rat heart [14]. Although resting cell length was reduced, the amplitude and time
course of myocyte shortening was not altered in ZDF rat compared to controls. Ventricular myocyte
shortening was associated with altered Ca++ transport. However, there was no significant difference in
the ratio of heart weight to body weight. A recent study in ZDF rats [24] suggests that diabetes per se
is not a critical factor in the induction of clinically significant cardiac dysfunction and some other
factors related to obesity might have greater impact on cardiac function. Previous studies in other
experimental models of diabetes, for example, streptozotocin-induced diabetic rats, have demonstrated
reduced weight gain which was variously associated with hyperglycemia, hypoinsulinemia, glycosuria,
depletion of body fat and liver glycogen [25,26]. A previous study demonstrated a prolonged time
course of myocyte shortening and relaxation of shortening in 9–13-week ZDF rats [13]. As in obese
humans, ZDF rats exhibit early β-cell compensation (hyperplasia) of insulin resistance followed by
decompensation (loss of cells) [27]. The early changes in β-cell responsiveness to glucose may
contribute to the hyperinsulinemia and subsequent insulin resistance [28]. ZDF rats also exhibit
reduced heart rate [29].
In type 2 diabetes and obesity metabolic disorders and inflammatory-linked pathologies, as seen in
ZDF rats, an increased production of reactive oxygen species, oxidative stress, altered cell signaling
and mitochondrial dysfunctions in heart and other tissues were observed [3,30–33]. We have also
confirmed increased oxidative stress due to increased production of ROS, resulting in increased
oxidative lipid and protein peroxidation in ZDF rat cardiac myocytes. Catalase appears to be the main
enzyme involved in H2O2 clearance as we observed no marked alteration in GSH-Px activity while
catalase activity was markedly increased in ZDF rats. Activation of catalase in ZDF rat heart might
reduce the pool of H2O2 in vivo, and hence disturb the balance of metabolism of this stable ROS which
might have implications in insulin signaling. There are reports which suggest that over expression of
antioxidant enzymes and altered H2O2 clearance in obesity may be responsible for the development of
insulin resistance and interfere in insulin-dependent signaling [34]. Activation of GSH metabolism by
GST enzyme and recycling of GSH by GSSG-reductase appears to be a defense response to prevent
the overquenching of intracellular ROS required for insulin signaling. H2O2 has also been shown to
modulate nitric oxide synthesis in cardiomyocytes [35]. Our study has clearly indicated that the
regeneration of GSH from GSSG significantly increased in ZDF rat heart due to an increase in
GSSG-reductase activity while no significant alteration in GSH-peroxidase (which utilizes GSSG)
activity was observed. This was accompanied by an increased GSH pool in cardiac myocytes. Our
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results thus suggest an induction of GSH-dependent antioxidant adaptive response in cardiac myocytes
in ZDF rat heart.
We observed an increased expression of CYP 2E1, iNOS, HO-1 accompanied by increased protein
carbonylation in ZDF rat heart suggesting increased oxidative stress. Our previous studies on
streptozotocin-induced chronic metabolic complications have also shown increased oxidative stress,
mitochondrial dysfunction and altered expression of CYP 2E1 and oxidative stress marker
proteins [16–18]. Increased expression of CYP 2E1 in oxidative stress conditions have also been
shown to induce apoptosis in cardiomyocytes [36]. Recently, it has also been shown that the inhibition
of protein carbonylation by antioxidants prevents the metabolic complications in ZDF rats [37].
Mitochondrial dysfunction, reduction in the activities of the respiratory complexes and reduced
expression of cytochrome c oxidase was also observed in ZDF rat heart. Altered oxygen consumption
in ZDF rat heart might be associated with compromised mitochondrial bioenergetics, which resulted in
the reduced activities of the respiratory complexes. Altered mitochondrial function and increased
oxidative stress in ZDF rats has consequences in modulating JNK and NF-κB dependent cell signaling.
We observed decreased JNK phosphorylation and reduced expression of IκB-α in ZDF rat heart. This
might suggest an increased inflammatory response in ventricular myocytes. A recent study has also
shown a decreased expression of p-JNK in transgenic type 2 diabetic rats which is responsible for
modulation of MAPK cascade as observed in diabetic cardiomyopathy [38]. JNK-dependent activation
of NF-κB in cardiomyocytes induced by hyperglycemia, inflammation and oxidative stress has also
been reported [39]. It has also been suggested that activation of JNK can be negatively regulated by
NF-κB inhibition [40]. Altered JNK-dependent signaling pathway, due to increased oxidative stress,
has also been reported in human islets and ZDF rats leading to the onset of mitochondrial dysfunction
in the diabetic islets [41]. Increased NF-κB activity and decrease in the inhibitory IκB-α expression
also suggest an increase in proinflammatory signaling in obesity [42]. Increased NF-κB activity as
seen in obesity and other inflammatory conditions is also involved in uncoupling insulin resistance
from lipid metabolism [43]. A study by Aragno et al. [44] has also shown reduced left ventricle
myocardial contractility and increased cardiomyopathy, following the impairment of NF-κB signaling
in ZDF rats. Our results may also suggest a crucial role of these signaling proteins in cardiomyocyte
survival from oxidative stress related proinflammatory responses.
3. Experimental Section
3.1. Chemicals
Cytochrome c, reduced and oxidized glutathione (GSH), 5,5'-dithio-bis(2-nitrobenzoic acid),
1-chloro 2,4-dinitrobenzene (CDNB), cumene hydroperoxide, glutathione reductase, thiobarbituric
acid, NADH, NADPH, coenzyme Q2, antimycin A, dodecyl maltoside, dimethylnitrosamine (DMNA),
dimethylphenylhydrazine (DNPH) were purchased from Sigma-Aldrich Fine Chemicals (St. Louis,
MO, USA). 2',7'-Dichlorofluorescein diacetate (DCFDA) was procured from Molecular Probes
(Eugene, OR, USA). Polyclonal antibodies against iNOS, CYP2E1, cytochrome c oxidase subunit 1,
JNK, IκB-α and β-actin were purchased from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA),
HO-1 from Abcam (Cambridge, MA, USA) and p-JNK from Cell Signaling Technology, Inc.
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(Danvers, MA, USA). Reagents for SDS-PAGE and Western blot analyses were purchased from Gibco
BRL (Grand Island, NY, USA) and Bio Rad Laboratories (Richmond, CA, USA).
3.2. Animal and Tissue Preparation
Experiments were performed in elderly (aged 30–34 weeks) Zucker diabetic fatty (ZDF; FA/FA)
rats (n = 4, average body wt = 485 g; average blood glucose = 478 mg/dL) and age-matched Zucker
lean (ZL; +/FA) controls (n = 5, average body wt = 400 g; average blood glucose = 108 mg/dL)
(Charles River Laboratories, UK). Approval for this project was obtained from the Animal Ethics
Research Committee, College of Medicine & Health Sciences, United Arab Emirates University and
all the animals were used according to the safe practice for animals in research guidelines as stipulated
by NIH, USA.
Left ventricle heart muscles were dissected from male ZDF and from age-matched ZL control
rats and rinsed with ice-cold saline. Isolated tissues were homogenized (10% w/v) in isotonic
100 mM potassium phosphate buffer (pH7.4) containing 1 mM EDTA and 0.1 mM
phenylmethylsulfonylfluoride (PMSF, a protease inhibitor). The homogenate was centrifuged at 1000g
for 10 min and supernatant was used for further analysis. Protein concentration was measured using
BioRad reagent as described before [15–18].
3.3. Measurement of ROS
Production of ROS in ZDF and ZL rat heart cellular fractions was measured using the DCFDA
fluorescence method as described before [18].
3.4. Protein and Lipid Peroxidation (LPO) and Catalase Activities
Protein peroxidation as a marker of increased oxidative stress was measured in ZDF and ZL rat
hearts by DNPH conjugation method as described before [18]. NADPH-dependent-membrane lipid
peroxidation was measured as malonedialdehyde formed using the standard thiobarbituric acid method
as described before [15]. Catalase activity was performed by the method of Beers and Sizer (1952) in
which the disappearance of peroxide was followed spectrophotometrically at 240 nm. One Unit
decomposes one micromole of hydrogen peroxide per minute at 25 °C and pH 7.0 under the specified
conditions [19].
3.5. Measurement of GSH-Redox Metabolism
GSH is the most important cellular antioxidant protecting tissues from oxidative insults. Alterations
in GSH-redox metabolism by GSH-peroxidase/reductase and transferases are the key indicators of
perturbed antioxidant metabolism. GSH concentration in the tissue homogenate, prepared as described
above, was measured by NADPH-dependent GSSG-reductase catalyzed conversion of oxidized GSSG
to GSH. Glutathione S-transferase (GST) activity using CDNB, glutathione peroxidase (GSH-Px)
activity using cumene hydroperoxide and glutathione-reductase activity using GSSG/NADPH as the
respective substrates were measured by standard protocols as described before [15,16].
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3.6. Measurement of CYP 2E1 Activity
CYP 2E1 enzyme activity was measured in left ventricle homogenate from the ZDF and ZL rat
heart using dimethylnitrosamine (DMNA) substrate in the presence of NADPH in the appropriate
buffer (pH 7.4) as described before [16].
3.7. Measurement of Mitochondrial Respiratory Enzyme Complexes
The freshly isolated heart muscle homogenate (5 μg protein) was suspended in 1.0 mL of 20 mM
KPi buffer, pH 7.4, in the presence of the detergent, lauryl maltoside (0.2%). NADH-ubiquinone
oxidoreductase (Complex I), and cytochrome c oxidase (Complex IV) activities were measured using
the substrates coenzyme Q2 and reduced cytochrome c, respectively, according to previously described
methods [18].
3.8. SDS-PAGE and Western Blot Analysis
Homogenates (50 μg protein) from ZDF and ZL rat hearts were electrophoretically separated by
12% SDS-PAGE [22] and transferred on to nitrocellulose paper [23]. The expression of specific
oxidative stress marker proteins (HO-1, iNOS, CYP2E1, Cyt c Ox) and cell signaling transcription
regulatory proteins (JNK, p-JNK and IκB-α) was checked by immunoreactions with their specific
antibodies by Western blot analysis as described before [15–18]. β-actin was used a loading control.
Densitometric analysis of the protein bands was performed using a gel documentation system (Vilber
Lourmat, France) and expressed as relative intensity (R.I) compared to the protein expression of ZL
which was arbitrarily taken as 1.0.
3.9. Statistical analysis
Values were calculated as mean ± S.E.M. of at least three determinations. Statistical significance of
the data was assessed using Student’s t-test and p-values ≤ 0.05 were considered significant.
4. Conclusions
Our results have demonstrated increased oxidative stress and mitochondrial dysfunctions in elderly
Zucker diabetic fatty rat hearts. The study has implications in elucidating the etiology and pathology of
cardio vascular complications in diabesity.
Acknowledgements
Authors wish to acknowledge the support of Terry Fox Cancer Research Fund and a Project Grant
from the College of Medicine and Health Sciences, U.A.E. University, U.A.E.
Conflict of Interest
The authors declare no conflict of interest.
Int. J. Mol. Sci. 2012, 13
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