Activated apoptotic and anti-survival effects
on rat hearts with fructose induced metabolic
syndrome
Shiu-Min Cheng1, Yu-Jung Cheng2, Liang-Yi Wu3, Chia-Hua Kuo2,4, Yi-Shin Lee2,
Ming-Che Wu5, Chih-Yang Huang6,7,8, Hua Ting5,9,†and Shin-Da Lee2,10,11*,†
1Department of Psychology, Asia University, Taichung, Taiwan
2Department of Physical Therapy, Graduate Institute of Rehabilitation Science, China
Medical University, Taichung, Taiwan
3Department of Bioscience Technology, Chung Yuan Christian University, Taoyuan, Taiwan 4Department of Sports Science, University of Taipei, Taipei, Taiwan
5Department of Physical Medicine and Rehabilitation, Chung-ShanMedicalUniversity
Hospital, Chung-Shan MedicalUniversity, Taichung, Taiwan
6School of Chinese Medicine, College of Chinese Medicine, China Medical University,
Taichung, Taiwan
7Graduate Institute of Basic Medical Science, China Medical University, Taichung, Taiwan 8Department of Health and Nutrition Biotechnology, Asia University, Taichung, Taiwan 9Institute of Medicine, Chung-Shan Medical University, Taichung, Taiwan
10Department of Healthcare Administration, Asia University, Taichung, Taiwan 11School of Rehabilitation Medicine, Shanghai University of TCM, Shanghai, China
Consumption of fructose has been linked to the development of metabolic syndrome, whereas the cardiomyopathic changes and cardiac apoptosis of dietary high-fructose intake have not yet been clarifed. The purpose of this study was to evaluate the effects of high-fructose on cardiac apoptotic and survival pathways. Thirty-two Wistar rats were randomly divided into a control group (CON), which received a standard chow diet, and a fructose-induced metabolic syndrome group (FIMS), which received a 50% fructose-content diet for 13 weeks. Histopathological analysis,
TUNEL assays and Western blotting were performed on the excised hearts fromboth groups. The blood pressure, glucose, insulin,
triglyceride and
cholesterol levelswere signifcantly increased in the FIMS group, compared with the CON group. The abnormalmyocardial
interstitial space and increased cardiac TUNEL-positive apoptotic cells were observed in the FIMS group. The TNF-a, TNF receptor 1, Fas
ligand, Fas receptor, FADD, and activated caspase-3 and 8 protein levels (Fas pathway) and the Bax, Bak, Bax/Bcl-2, Bak/Bcl-xL, cytosolic
cytochrome c, and activated caspase-3 and nine protein levels (mitochondria pathway) were increased in the FIMS group compared with those
in the CON group. The IGFI, IGFI-R, p-PI3K, p-Akt, Bcl-2 and Bcl-xL protein levels (survival pathway) were all signifcantly decreased in the FIMS group compared with those in the CON group.
High-fructose intake elevated blood pressure and glucose levels; moreover, high-fructose
diet activated cardiac Fas-dependent and mitochondria-dependent apoptotic pathways and suppressed the survival pathway, which might provide
one possible mechanism for developing heart failure in patients with metabolic syndrome. Copyright © 2013 John Wiley & Sons, Ltd. key words—Apoptosis; Fructose; Fas receptor; Heart; Metabolic syndrome;
TNF-alpha INTRODUCTION
Metabolic syndrome is a cluster of symptoms including central obesity, insulin resistance, hyperglycemia, dyslipidemia
and hypertension, all of which are risk factors for the development of obesity, type 2 diabetes and cardiovascular disease
(CVD).1–4 The high prevalence of metabolic syndrome has signifcant public health implications because of the sixfold risk of developing type 2 diabetes, twofold increased risk of prevalence of coronary heart disease and threefold increased risk of mortality attributable to coronary heart disease.5 Most studies regarding metabolic syndrome show that it may lead to deteriorated cardiac geometry and function of CVD and heart failure.2–5 Cellular apoptosis in terminally differentiated cardiomyocytes is a very critical pathological
mechanism in the cause of heart failure, whereas, on the other hand, the process of apoptotic interruption may be allowed to develop a novel strategy to reverse or attenuate heart failure.6,7 Moreover, cardiac apoptosis has been found in obesity, diabetes and hypertension.7–10 Apoptosis, a physiological program of cellular death, may contribute to many cardiac disorders.11,12 The
occurrence of apoptosis has been reported to contribute to the loss of cardiomyocytes in cardiomyopathy and is recognized as a
predictor of adverse outcomes in subjects with cardiac diseases or heart failure.6 The ‘extrinsic’ Fas ligand or tumor necrosis factor-alpha (TNF-a)–dependent (type I) apoptotic pathway is believed to be one of the major pathways directly to trigger cardiac
apoptosis.12–14 This pathway is often initiated by binding the Fas ligand to the Fas death receptor or by binding the TNF-a to TNF receptor 1 (TNFR1), which
results in the clustering of receptors and the initiation of an extrinsic pathway.13 Fas ligand and Fas receptor or TNF-a and
TNFR1 complex are known to lead to the formation of a death-inducing signal complex starting with the recruitment of the Fas-associated death domain (FADD) of the adaptor protein.13 FADD is known to function as a common signaling conduit in Fas and TNF-a–mediated apoptosis.15 FADD
recruits and aggregates the pro-caspase-8 and leads to its activation of caspase-8.14 The activated caspase-8 cleaves pro-caspase-3, which then undergoes autocatalysis to form active caspase-3, a principle effector caspase of apoptosis.16 The ‘intrinsic’ mitochondria-dependent (type II) apoptotic pathway starts from within the cell, resulting in the release of a number of pro-apoptotic factors from the intermembrane space of mitochondria.13,14 The mitochondria is the main site of action formembers of the apoptosis-regulating protein family exemplifed by Bcl-2 family, such as Bax (Bcl-2-associated X protein) and Bak (pro-apoptotic molecular).13
Commitment to apoptosis is typically governed by opposing factions of the Bcl-2 family, including pro-apoptotic versus
anti-apoptotic family members.17 Pro-apoptotic and prosurvival Bcl-2 family members can homodimerize or
neutralize each other so that the relative balance of these effectors strongly infuences cytochrome c release.18 Bcl-2 and
Bcl-xL, pro-survival proteins prevent cytochrome c release, whereas Bax and Bak enhance cytochrome c release from the mitochondria.13 When cytochrome c is released from mitochondria
into the cytosol, it is responsible for activating
caspase-9, which further activates caspase-3 and executes the apoptotic program.19 Cardiac Fas-dependent and
mitochondria-dependent apoptotic pathways are involved in many pathologic conditions such as hypoxic stress, hypertension and obesity.20–24 However, it is unclear whether cardiac
Fas-dependent and mitochondria-dependent apoptotic pathways mediate metabolic syndrome-related cardiac apoptosis.
Insulin-like growth factor I (IGFI) signaling is reported to contribute to the modulation of survival responses in cardiac tissues. Phosphatidylinositol 3-kinase (PI3K) and protein kinase B (Akt) are key signaling factors in insulin and IGFI-receptor (IGFI-R).25–28 Impaired IGFI signaling may contribute, at least partially, to the development of diabetes and the pathology of cardiac apoptosis in diabetic animals
and humans.8,29 Akt is one of the major upstream signal proteins of the Bcl-2 family, and phosphorylated Akt (p-Akt)
appears to promote the pro-survival pathway.30 However, the cardiac IGFI-related survival pathway in the metabolic syndrome animal model has not yet been reported.
A well-known experimental model ofmetabolic syndrome is induced by feeding rats with a high-fructose diet. This model
induces weight gain, hypertension, hyperinsulinemia, hyperlipidemia and insulin resistance.31,32 The current study was undertaken
to understand the effects of high-fructose on the cardiac
Fas-dependent apoptotic (TNF-a, TNFR1, Fas ligand, Fas receptor, FADD, activated 8, and activated
caspase-3), mitochondria-dependent apoptotic (Bax, Bak, cytosolic cytochrome
c, activated caspase-9 and activated caspase-3) and
survival (IGFI, IGFI-R, p-PI3K, p-Akt, Bcl-2 and Bcl-xL) pathways in rats. We hypothesized that fructose-induced metabolic
syndrome rats may be predisposed to more activated cardiac Fas-dependent and mitochondria-dependent apoptotic pathways, as well as suppressed cardiac IGFI-related survival and
Bcl-2 family associated pro-survival pathways. METHODS
Animals and induction of metabolic syndrome
Thirty-two male Wistar sixteen-week-old rats were obtained from the National Laboratory Animal Center, Taiwan.
Ambient temperature was maintained at 25 C, and the animals were kept on an artifcial 12-h light–dark cycle. The light
period beginning at 700 h. Rats were fed with a standard Purina chow diet (#5001, Purina, St. Louis, MO, USA; based on dry weight, composed of 23% protein, 56% carbohydrate, 4.5% fat, and 6%fber) and water ad libitum. All experimental procedures were performed according to the NIH Guide for the Care and Use of Laboratory Animals, and all protocols were approved by the Institutional Animal Care and Use Committee of China Medical University, Taichung, Taiwan. All animals were allowed to adapt to the environment for one week after their arrival before the experiment started. The animals were fed with standard Purina chow diet for 5 days, and no statistical differences in body weight, blood pressure, blood glucose, insulin, triglyceride, and cholesterol were found before the beginning of the experiment.
The glucose level was detected by Accu Soft (Roche, Indianapolis, IN, USA) test strips. Systolic, diastolic and mean arterial
blood pressure were measured with an automated tailcuff system (29SSP; IITC/Life Science Instruments). The average of fve consecutive readings for accurate measurement was used for blood pressure.
The animals were divided into a control group (CON, n = 16), which received standard Purina chow diet and a fructose-induced metabolic syndrome group (FIMS, n = 16), which received a high fructose-content diet (composed of
21% protein, 50% fructose, 5% fat and 8% fber as a percentage of total calories) . All groups were followed for 13 weeks.
Eight rats from each group were used for western blot analysis, and the remaining eight rats from each group were used
for pathological staining.
Blood collection and tissue extraction
At the end of the experiment, all animals were sacrifced after overnight fasting, trunk blood samples were collected in heparinized tubes. Blood samples were centrifuged at 2000 g for 10 min at 4 C; then, the plasma was collected and stored at 20 C until assayed for insulin, triglyceride and cholesterol. Cardiac tissue extracts were obtained by
homogenizing the left ventricle samples in a lysis buffer (20 mM of Tris, 2 mM of EDTA, 50 mM of
2-mercaptoethanol, 10% glycerol, pH 74, proteinase inhibitor (Roche), phosphatase inhibitor cocktail (Sigma Chemical Co., Louis, MO, USA)) at a ratio of 100 mg tissue per 1 ml buffer for 1 min. The homogenates were placed on ice for 10 min and then centrifuged at 12 000 g for 40 min, twice. The supernatant was collected and stored at 70 C for further investigations.
Cardiac characteristics and heart weight
The hearts of the rats from both groups were analysed using heart weight index and Western blotting. The hearts were excised and cleaned with phosphate-buffered saline (PBS). The left ventricles were separated and weighed. The ratios of the whole heart weight (WHW) to body weight (BW), the ratios of the left ventricular weight (LVW) to BW, the ratios of the LVW to WHW, the ratios of the WHW to tibia length and the ratios of the LVW to tibia length were calculated. Biochemical assays
Plasma insulin concentrations were measured using a commercial ELISA kit (Mercodia, Uppsala, Sweden). Plasma triglyceride and cholesterol concentrations were assayed with commercial kits (E. Merck, Darmstadt, Germany) by enzymatic photocolorimetric methods. Hematoxylin–eosin staining
The hearts from rats were excised and soaked in formalin, dehydrated through graded alcohols and embedded in paraffin wax. The 3-mm thick paraffin sections were cut from the
paraffin-embedded tissue blocks. The tissue sections were deparaffinized by immersing in xylene and rehydrated. They
were passed through a series of graded alcohols (100%, 95% and 75%), for 15 min of each. The slides were then dyed with hematoxylin for 5–10 min, followed by washing with tap water for 10–20 min. The slides were then soaked in mild warm water until it turned bright violent before putting it into eosin solution for 3–5 min. After gently rinsing with water, each slide was soaked in 85% alcohol and 100% alcohol I and alcohol II for 15 min each. The fnal step was to soak in xylene I and xylene II. Photomicrographs were obtained using Zeiss Axiophot microscopes. The slices were measured by the software ‘Adobe Photoshop CS3’. The mean number of myocardial interstitial spaces was quantifed for at least 5–6 separate felds2 slides3
regions of each left ventricle (upper,middle and lower) excised from the rats’ hearts. All counts were performed by at least two independent individuals in a blinded manner.8
DAPI staining and terminal deoxynucleotide transferasemediated dUTP nick end labeling.
The hearts were excised, soaked in formalin, dehydrated through graded alcohols and embedded in paraffin wax.
Sections 3 mm thick were prepared from these paraffinembedded tissue blocks. The sections were deparaffinized by immersing in xylene, rehydrated and incubated in phosphate-buffered saline with 2% H2O2 to inactivate endogenous
peroxidases. Next, the sections were incubated with proteinase K (20 mgml–1), washed in phosphate-buffered
saline and incubated with terminal deoxynucleotidyl transferase and fuorescein isothiocyanate-dUTP for 1 h at
37 C using an apoptosis detection kit (Roche). After washing twice in PBS, the sections were stained with 4’,
6-diamidine-2-phenylindole dihydrochloride (DAPI, Sigma Chemical Co.) for 5 min to allow the cell nucleus to be detected by UV light microscopic observations (blue). TUNEL-positive nuclei (fragmented DNA) fuoresce bright green at 450–500 nm, whereas DAPI-positive nuclei (intact DNA) fuoresce blue at 360 nm. The mean number of
TUNEL-positive cells were counted for at least 5–6 separate felds2 slides3 regions of each left ventricle (upper,
middle and lower) excised from the rats’ hearts. All
counts were performed by at least two independent individuals in a blinded manner.
Separation of cytosolic and mitochondrial fractions
To detect cytosolic cytochrome c, tissues were suspended in a buffer (50mM Tris (pH 75), 05 M NaCl, 10 mM of
EDTA (pH 75), 10% glycerol and proteinase inhibitor cocktail tablet (Roche) for 3 min on ice), homogenized by 40 strokes in a Dounce homogenizer, and centrifuged at
12 000 g for 15 min. The supernatant was the cytosol fraction, and the pellet was resuspended in lysis buffer as the
membrane fraction.
Electrophoresis and Western blot
Protein concentration of cardiac tissue extracts was determined by the Lowry protein assay. Protein samples (40 mg lane–1)
were separated on a 10% SDS polyacrylamide gel electrophoresis (SDS-PAGE) with a constant voltage of 75 V.
Electrophoresed proteins were transferred to a polyvinylidene difuoride (PVDF) membrane (045 mm pore size, Millipore, Bedford, MA, USA) with a transfer apparatus (Bio-Rad Laboratories Inc., Berkeley, CA, USA). PVDF membranes were incubated in 5% milk in TBS buffer. Primary antibodies including TNF-a, TNFR1, Fas ligand, Fas receptor, FADD, Bax, Bak, Bcl-2, Bcl-xL, caspase-3, caspase-8, caspase-9, cytochrome c, IGFI, IGFI-R, p-PI3K (Santa Cruz Biotechnology, Santa Cruz, CA, USA), p-Akt (Cell Signaling Technology
Inc., Beverly, MA, USA) and a-tubulin (Neo Markers,
Fremont, CA, USA)were diluted to 1:500 in antibody binding buffer overnight at 4 C. The immunoblots were washed three times in TBS buffer for 10min and then immersed in a second antibody solution containing goat anti-mouse IgG-HRP, goat anti-rabbit IgG-HRP, or donkey anti goat IgG-HRP
(Santa Cruz Biotechnology) for 1 h and diluted 500-fold in TBS buffer. The immunoblots were then washed three
times in TBS buffer for 10 min. The immunoblotted proteins were visualized using an enhanced chemiluminescence
ECL Western blotting luminal reagent (Santa Cruz
Biotechnology) and quantifed using a Fujiflm LAS-3000
chemiluminescence detection system (Fujiflm, Tokyo, Japan). Densitometric analyses of immunoblots were
performed with an AlphaImager 2200 digital imaging system (Digital Imaging System, San Leandro, CA, USA).
Statistical analysis
All data for body weight, heart weight index, blood pressure, biochemical parameters, protein levels, and percentage of TUNEL-positive cells were compared between the CON and FIMS groups using Student’s t-test for two independent samples. In all cases, a difference at P<005 was considered statistically signifcant.
RESULTS
Cardiac characteristics, blood pressure and biochemical parameters
The index of whole heart weight (WHW), left ventricular weight (LVW), WHW/body weight (BW), LVW/BW,
LVW/WHW and WHW/tibia length were similar in the CON and FIMS groups. The systolic blood pressure (SBP),
diastolic blood pressure (DBP) and mean arterial blood pressure (MBP) in the FIMS group were higher than those in the
CON group (P<005), as shown in Table 1. The concentrations of blood glucose, plasma insulin, triglyceride and cholesterol in the FIMS group were higher than those in the
CON group (Table 1).
Cardiac histopathological changes
To investigate changes of cardiac architecture in fructoseinduced metabolic syndrome, we performed a histopathological analysis of ventricular tissue stained with hematoxylin and eosin (H&E). The ventricular myocardium in the CON
group showed normal architecture with normal interstitial space, but the abnormal myocardial architecture and the increased interstitial spaces were observed in the FIMS group (Figure 1A and 1B).
TUNEL-positive apoptotic cells of cardiac tissue
To reconfrm the apoptotic activity in fructose-induced metabolic syndrome, we examined the apoptotic cardiac cells in
the excised hearts of the CON and FIMS groups by TUNEL assay. We observed that the left ventricles stained with TUNEL assay showed signifcantly increased TUNELpositive cardiac cells in the FIMS group compared with the
CON group, at 400 magnifcation images (Figure 1C and D). Upstream components of cardiac Fas-dependent apoptotic pathways
To understand the changes to the cardiac Fas-dependent apoptotic pathway in fructose-induced metabolic syndrome, the protein levels of TNF-a, TNFR1, Fas ligand, Fas receptor and FADD were measured in the excised hearts of the CON and FIMS groups by Western blotting (Figure 2A). The protein levels of TNF-a, TNFR1, Fas ligand, Fas receptor and FADD in the FIMS group were signifcantly higher
than those in the CON group (Figure 2B).
Upstream components of cardiac mitochondria-dependent apoptotic and Bcl-2 family–associated pro-survival pathways
To understand the changes of cardiac mitochondriadependent apoptotic and Bcl-2 family associated prosurvival
pathways in fructose-induced metabolic syndrome,
the protein levels of Bax, Bak, Bcl-2, Bcl-xL and cytosolic cytochrome
c were measured in the excised hearts of the CON
and FIMS groups using Western blotting (Figure 3A). The protein levels of Bax, Bak, cytochrome c, Bax/Bcl-2 and Bak/Bcl-xL in the FIMS group were signifcantly increased
compared with those in the CON group (Figure 4B). In addition, the protein levels of Bcl-2 and Bcl-xL were signifcantly
decreased in the FIMSgroup compared with those in the CON group (Figure 3B).
Downstream components of cardiac Fas-dependent and mitochondria-dependent apoptotic pathways
To identify the downstream components of cardiac Fas (caspase-8 and 3) and mitochondria (caspase-9 and 3)–
dependent apoptotic pathways in fructose-induced metabolic syndrome, the protein levels of activated caspase-8, 9 and 3 were measured in the excised hearts of the CON and FIMS
groups usingWestern blotting (Figure 4A). The protein levels of caspase-8, 9 and 3 were signifcantly increased in the FIMS group compared with those in the CON group (Figure 4B). Cardiac survival pathway
To identify the cardiac IGFI-R-related PI3K-Akt survival pathway in fructose-induced metabolic syndrome, the protein levels of IGFI, IGFI-R, p-PI3K, and p-Akt were measured in the excised hearts of the CON and FIMS groups using Western blotting (Figure 5A). The protein levels of IGFI, IGFI-R, p-PI3K and p-Akt were signifcantly decreased in the FIMS group compared with those in the CON group (Figure 5B).
DISCUSSION
Our main new fndings can be summarized as follows: (1) The blood pressure, glucose, insulin, triglyceride and
cholesterol levels were signifcantly increased in the fructose (50%)–fed rats, a metabolic syndrome model, relative to the non-fructose fed rats. (2) Abnormal myocardial architecture, enlarged interstitial space and increased cardiac TUNELpositive apoptotic cells were observed in the fructoseinduced
metabolic syndrome but not in the non-metabolic
syndrome. (3) The cardiac Fas-dependent apoptotic proteins (TNF-a, TNFR1, Fas ligand, Fas receptor, FADD, activated
caspase-8 and activated caspase-3) in the fructose-induced metabolic syndrome were signifcantly increased compared to the non-metabolic syndrome. (4) The cardiac
mitochondria-dependent apoptotic proteins (Bax, Bak, Bax/ Bcl-2, Bak/Bcl-xL, cytosolic cytochrome c, activated
caspase-9 and activated caspase-3) in the fructose-induced metabolic syndrome were signifcantly increased compared with the non-metabolic syndrome. (5) Cardiac IGFI-related
survival proteins (IGFI, IGFI-R, p-PI3K and p-Akt) and Bcl-2 family–associated pro-survival proteins (Bcl-2 and Bcl-xL) in the fructose-induced metabolic syndrome were signifcantly
decreased compared with the non-metabolic syndrome. After integrating our current fndings into previously
proposed theories, a hypothesized diagram is drawn in Figure 6, which suggests that cardiac Fas receptor-dependent
and mitochondria-dependent pathways were increased, whereas cardiac IGFI-R/PI3K/Akt survival and Bcl-2 family
associated pro-survival pathways were decreased in the fructose feeding induced metabolic syndrome.
The consumption of fructose has increased, largely
because of an increased consumption of soft drinks and many juice beverages containing sucrose (table sugar consists of 50% fructose, 50% glucose) or high-fructose corn syrup (a single can of beverage contains about 42%–55%
fructose).32,33 Dietary high-fructose intake has been suggested to be an important factor contributing to the development of
symptoms of metabolic syndrome. Recent evidence suggests
that fructose feeding in rats develops the features of the metabolic syndrome model in many of the same pathophysiological
defcits as noted in metabolic syndrome humans, such as insulin resistance, weight gain, hyperlipidemia, hyperinsulinemia,
hypertriacylglycerolemia, impaired glucose tolerance, hypertension, myocardial functional abnormalities and heart
failure.3,4,31,32,34 In the current study, daily 50% fructose feeding for 13 weeks caused increases in blood pressure, glucose,
insulin, triglyceride and cholesterol levels. Furthermore, abnormal myocardial architecture, enlarged interstitial space and
increased TUNEL-positive apoptotic cardiac cells were
observed in the rat hearts. The physiological parameters and cardiomyopathic changes found in the current animal models may provide one mechanism to clarify how cardiac morphological change occurs in metabolic syndrome humans.
The Fas receptor-dependent apoptotic pathway is mediated by Fas ligand, Fas receptor, TNF-a, TNFR1, FADD and activated caspase-8. The mitochondria-dependent apoptotic pathway
is mediated by Bax, Bak, cytochrome c, activated
caspase-9 and activated caspase-3.12,13 In the current study, 50% fructose-fed rats appeared to have signifcantly activated Fas receptor-dependent apoptotic pathway, as evidenced by increases
in Fas ligand, Fas receptor, TNF-a, TNFR1, FADD, activated caspase-8 and activated caspase-3 levels in the hearts.
Additionally, fructose-fed rats appeared to have a signifcantly activated mitochondria-dependent apoptotic pathway, as evidenced by increases in the levels of Bax, Bak, Bax/Bcl-2, Bak/Bcl-xL, cytochrome c, activated caspase-9 and activated caspase-3 in the hearts. All key components of Fas
receptordependent
and mitochondria-dependent apoptotic pathways,
from upstream cascade to downstream cascade, consistently show pro-apoptotic effects in the hearts excised from the fructose-induced metabolic syndrome rats. These are the frst data to demonstrate that fructose intake activates the cardiac Fas receptor-dependent and mitochondria-dependent apoptotic pathways, which might lead to cardiac apoptosis.
The IGFI/IGFI-R and their downstream PI3K and Akt signaling pathways have been indicated to contribute to modulation of survival and apoptotic responses in cardiac tissue.25,35 Our previous study shows decreased IGFI/PI3K/Akt survival pathways via decreases in IGFI, IGFI-R, PI3K and Akt in the
diabetic rat heart.8 In the present study, the cardiac IGFIrelated survival signaling was signifcantly decreased in 50%
fructose-fed rats based on decreased IGFI, IGFI-R, p-PI3K
and p-Akt levels. The mitochondria-dependent apoptotic pathway is tightly controlled by the Bcl-2 family. Pro-apoptotic
and pro-survival members of the Bcl-2 family seem to interact with and neutralize each other so that the relative balance of these effectors strongly infuences cell fate.18,36 Shifting the balance of Bcl-2 family members toward pro-apoptotic effects will activate caspase-9, which further activates caspase-3 and executes the apoptotic program.19 In the current study, the cardiac pro-survival pathway was signifcantly decreased in 50% fructose-fed rats based on the decreased Bcl-2 and
Bcl-xL levels. Therefore, our fndings strongly suggest that the cardiac IGFI-related survival and Bcl-2 family associated
pro-survival pathways become less activated in the fructoseinduced metabolic syndrome animal model, which might lead
to developing cardiac apoptosis and the consequential development of heart failure.
HYPOTHESIZED AND CLINICAL APPLICATION
An increase in fructose intake has been linked with a rise in obesity and metabolic disorders. The current metabolic syndrome animal model under high fructose-feeding proves to be an important representation of metabolic syndrome inducing abnormal myocardial architecture and cardiac
apoptosis in metabolic syndrome humans because of cardiac tissues being difficult to sample from human hearts; moreover, high-fructose consumption increased blood pressure, glucose, insulin, triglyceride and cholesterol levels. Previous studies indicated that obesity and hypertension enhanced cardiac Fasdependent
and mitochondria-dependent apoptotic pathways,
9,10,23,24 and streptozotocin-induced diabetes activated cardiac mitochondria-dependent apoptotic pathways in rat
models.8,35 Overall, our current fndings indicate that the activation of cardiac Fas-dependent and mitochondria-dependent apoptotic pathways, as well as the cardiac IGFI/PI3K/Akt survival
pathway being suppressed in fructose-induced metabolic syndrome rats, might provide an important mechanism in the explanation of the development of cardiomyopathy.
Furthermore, additional questions will be raised, such as whether anti-apoptotic therapy might be benefcial to attenuate cardiac Fas-dependent and/or mitochondria-dependent apoptotic pathways when considering possible therapeutic agents to control
or prevent the development of apoptosis-related cardiac diseases in metabolic syndrome. Of course, further therapeutic
and clinical studies are required to clarify the effects of treatments or the survival and apoptotic mechanisms in metabolic
CONFLICT OF INTEREST
The authors have declared that there is no confict of interest. ACKNOWLEDGMENT
This study was supported by grants from the National Science Council (NSC 99-2410-H-468-030-MY3 and
98-2314-B-040-001-MY3) and the China Medical University and Asia University (CMU99-ASIA-05) in Taiwan. This study is
supported in part by Taiwan Department of Health Clinical Trial and Research Center of Excellence (DOH101-TD-B-111-004). The pathway diagram in the current study was modifed from the Pathway Central from SABiosciences.
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