Doxorubicin-Induced Cardiomyopathy: A Preliminary Study on the Cardioprotective Benefits of 7-Hydroxyflavanone.

The therapeutic properties of flavonoids are reported to offer cardioprotective benefits against doxorubicin (Dox)-induced cardiotoxicity (DIC). In the current study, we aimed to investigate the prophylactic properties of 7-hydroxyflavanone (7H), a flavonoid with antioxidative properties, against DIC. An in vitro model of DIC was established by exposing H9c2 cardiomyoblasts to Dox for 6 days. Similarly, cells were also co-treated with 7H to assess its ability to mitigate DIC. The data obtained indicate that 7H, as a co-treatment, alleviates Dox-induced oxidative stress by enhancing total glutathione content (p ≤ 0.001) and superoxide dismutase activity (p ≤ 0.001) whilst decreasing ROS (p ≤ 0.001), malondialdehyde production (p ≤ 0.001) and the secretion of interleukin-6 (p ≤ 0.001). The data also showed an improvement in mitochondrial function as shown via enhanced bioenergetics, mitochondrial membrane potential, and PGC1-alpha (p ≤ 0.05) and pAMPK (p ≤ 0.001) expression. The cardioprotective potential of 7H was further highlighted by its ability attenuate Dox-induced caspase 3/7 activity (p ≤ 0.001), apoptosis (p ≤ 0.001) and necrosis (p ≤ 0.05). In conclusion, our findings demonstrated the cardioprotective benefits of 7H and thus suggests that it could be a suitable candidate cardioprotective agent against DIC.

Prevention of Anthracycline-Induced Cardiotoxicity: The Good and Bad of Current and Alternative Therapies.

Doxorubicin (Dox)-induced cardiotoxicity (DIC) remains a serious health burden, especially in developing countries. Unfortunately, the high cost of current preventative strategies has marginalized numerous cancer patients because of socio-economic factors. In addition, the efficacy of these strategies, without reducing the chemotherapeutic properties of Dox, is frequently questioned. These limitations have widened the gap and necessity for alternative medicines, like flavonoids, to be investigated. However, new therapeutics may also present their own shortcomings, ruling out the idea of "natural is safe". The U.S. Food and Drug Administration (FDA) has stipulated that the concept of drug-safety be considered in all pre-clinical and clinical studies, to explore the pharmacokinetics and potential interactions of the drugs being investigated. As such our studies on flavonoids, as cardio-protectants against DIC, have been centered around cardiac and cancer models, to ensure that the efficacy of Dox is preserved. Our findings thus far suggest that flavonoids of Galenia africana could be suitable candidates for the prevention of DIC. However, this still requires further investigation, which would focus on drug-interactions as well as in vivo experimental models to determine the extent of cardioprotection.

Molecular insights into the pathophysiology of doxorubicin-induced cardiotoxicity: a graphical representation.

A breakthrough in oncology research was the discovery of doxorubicin (Dox) in the 1960's. Unlike other chemotherapy drugs, Dox was determined to have a greater therapeutic index. Since its discovery, Dox has, in part, contributed to the 5-10-year survival increase in cancer patient outcomes. Unfortunately, despite its efficacy, both in adult and pediatric cancers, the clinical significance of Dox is tainted by its adverse side effects, which usually manifest as cardiotoxicity. The issue stems from Dox's lack of specificity which prevents it from accurately distinguishing between cancer cells and healthy cell lines, like cardiomyocytes. In addition, the high binding affinity of Dox to topoisomerases, which are abundantly found in cancer and cardiac cells in different isoforms, potentiates DNA damage. In both cell lines, Dox induces cytotoxicity by stimulating the production of pro-oxidants whilst inhibiting antioxidant enzymatic activity. Given that the cardiac muscle has an inherently low antioxidant capacity makes it susceptible to oxidative damage thereby, allowing the accumulation of Dox within the myocardium. Subsequently, Dox drives the activation of cell death pathways, such as ferroptosis, necroptosis and apoptosis by triggering numerous cellular responses that have been implicated in diseases. To date, the exact mechanism by which Dox induces the cardiotoxicity remains an aspect of much interest in cardio-oncology research. Hence, the current review summarizes the proposed mechanisms that are associated with the onset and progression of DIC.

Cardioprotective Function of Green Rooibos (Aspalathus linearis) Extract Supplementation in Ex Vivo Ischemic Prediabetic Rat Hearts.

Diabetic patients develop ischemic heart disease and strokes more readily. Following an ischemic event, restoration of blood flow increases oxidative stress resulting in myocardial damage, termed ischemia/reperfusion injury. Aspalathus linearis (rooibos), rich in the antioxidant phenolic compound aspalathin, has been implicated as cardioprotective against ischemia/reperfusion injury with undefined mechanism in control rats. Primarily, the therapeutic potential of Afriplex green rooibos extract to prevent ischemia/reperfusion injury in cardiovascular disease-compromised rats was investigated. Additionally, Afriplex Green rooibos extract's cardioprotective signaling on metabolic markers and stress markers was determined using western blotting. Three hundred male Wistar rats received either 16-wk standard diet or high-caloric diet. During the final 6 wk, half received 60 mg/kg/day Afriplex green rooibos extract, containing 12.48% aspalathin. High-caloric diet increased body weight, body fat, fasting serum triglycerides, and homeostatic model assessment of insulin resistance - indicative of prediabetes. High-caloric diet rats had increased heart mass, infarct size, and decreased heart function. Afriplex green rooibos extract treatment for 6 wk lowered pre-ischemic heart rate, reduced infarct size, and improved heart function pre- and post-ischemia, without significantly affecting biometric parameters. Stabilized high-caloric diet hearts had decreased insulin independence via adenosine monophosphate activated kinase and increased inflammation (p38 mitogen-activated protein kinase), whereas Afriplex green rooibos extract treatment decreased insulin dependence (protein kinase B) and conferred anti-inflammatory effect. After 20 min ischemia, high-caloric diet hearts had upregulated ataxia-telangiectasia mutated kinase decreased insulin independence, and downregulated insulin dependence and glycogen synthase kinase 3 ß inhibition. In contrast, Afriplex green rooibos extract supplementation downregulated insulin independence and inhibited extracellular signal-regulated kinase 1 and 2. During reperfusion, all protective signaling was decreased in high-caloric diet, while Afriplex green rooibos extract supplementation reduced oxidative stress (c-Jun N-terminal kinases 1 and 2) and inflammation. Taken together, Afriplex green rooibos extract supplementation for 6 wk preconditioned cardiovascular disease-compromised rat hearts against ischemia/reperfusion injury by lowering inflammation, oxidative stress, and heart rate.

Ataxia Telangiectasia Mutated Protein Kinase: A Potential Master Puppeteer of Oxidative Stress-Induced Metabolic Recycling.

Ataxia Telangiectasia Mutated protein kinase (ATM) has recently come to the fore as a regulatory protein fulfilling many roles in the fine balancing act of metabolic homeostasis. Best known for its role as a transducer of DNA damage repair, the activity of ATM in the cytosol is enjoying increasing attention, where it plays a central role in general cellular recycling (macroautophagy) as well as the targeted clearance (selective autophagy) of damaged mitochondria and peroxisomes in response to oxidative stress, independently of the DNA damage response. The importance of ATM activation by oxidative stress has also recently been highlighted in the clearance of protein aggregates, where the expression of a functional ATM construct that cannot be activated by oxidative stress resulted in widespread accumulation of protein aggregates. This review will discuss the role of ATM in general autophagy, mitophagy, and pexophagy as well as aggrephagy and crosstalk between oxidative stress as an activator of ATM and its potential role as a master regulator of these processes.

The modulating effects of green rooibos (Aspalathus Linearis) extract on vascular function and antioxidant status in obese Wistar rats.

PURPOSE: Obesity is associated with the development of risk factors for cardiovascular disease (CVD) and polyphenols have been shown to possess ameliorative effects against obesity-induced CVD risk factors. Rooibos (Aspalathus linearis) is rich in polyphenols, therefore we investigated the cardioprotective effects of aspalathin-rich green rooibos (GRT) on obesity-induced CVD risk factors in obese Wistar rats. METHODS: Adult male Wistar rats (n = 20 per group) were fed a control or a high-fat diet (HFD) for 16 weeks and treated with GRT (60 mg/kg/day) for six weeks. Blood pressure was monitored throughout. Vascular reactivity was measured and Western blots of cell-signalling proteins (eNOS, AMPK and PKB) were performed in aortic tissues. Effects on oxidative stress were determined by measuring antioxidant enzyme activity and thiobarbituric reactive substance (TBARS) levels in the liver. RESULTS: HFD animals had (1) increased blood pressure, (2) impaired vasodilation, (3) attenuated PKB and AMPK expression, (4) decreased antioxidant enzyme activity, (5) increased malondialdehyde (MDA) levels, and (6) increased phosphorylated eNOS levels. Treatment with GRT extract significantly alleviated these obesity-induced CVD risk factors. CONCLUSIONS: Supplementation with GRT extract alleviated cardiovascular risk factors in the HFD animals, suggesting a therapeutic potential for GRT in obesity-induced cardiovascular risk.

The Prophylactic Effect of Pinocembrin Against Doxorubicin-Induced Cardiotoxicity in an In Vitro H9c2 Cell Model.

BACKGROUND: The clinical use of Doxorubicin (Dox) is significantly limited by its dose-dependent cardiotoxic side effect. Accumulative evidence suggests that the use of flavonoids, such as the antioxidative Pinocembrin (Pin), could be effective in the prevention of Dox-induced cardiotoxicity. Accordingly, we investigated the ability of pinocembrin (Pin) to attenuate Dox-induced cardiotoxicity in an in vitro H9c2 cardiomyoblast model. METHODOLOGY: The cardioprotective potential of Pin was established in H9c2 cells. Here, cells were treated with Dox (2µM), Dox (2µM) + Pin (1µM), and Dox (2µM) + Dexrazoxane (20µM) for 6 days. Thereafter, the safe co-administration of Pin with Dox, in a cancer environment, was investigated in MCF-7 breast cancer cells subjected to the same experimental conditions. Untreated cells served as the control. Subsequently, Pin's ability to attenuate Dox-mediated oxidative stress, impaired mitochondrial bioenergetics and potential, as well as aggravated apoptosis was quantified using biochemical assays. RESULTS: The results demonstrated that co-treatment with Pin mitigates Dox-induced oxidative stress by alleviating the antioxidant enzyme activity of the H9c2 cells. Pin further reduced the rate of apoptosis and necrosis inferred by Dox by improving mitochondrial bioenergetics. Interestingly, Pin did not decrease the efficacy of Dox but, rather increased the rate of apoptosis and necrosis in Dox-treated MCF-7 cells. CONCLUSION: The findings presented in this study showed, for the first time, that Pin attenuates Dox-induced cardiotoxicity without reducing its chemotherapeutic effect. We propose that additional studies, using in vivo models, should be conducted to further investigate Pin as a suitable candidate in the prevention of the cardiovascular dysfunction inferred by Dox administration.

Mitochondrial Oxidative Phosphorylation Function and Mitophagy in Ischaemic/Reperfused Hearts from Control and High-Fat Diet Rats: Effects of Long-Term Melatonin Treatment.

PURPOSE: Oxidative stress causes mitochondrial dysfunction in myocardial ischaemia/reperfusion (I/R) as well as in obesity. Mitochondrial depolarization triggers mitophagy to degrade damaged mitochondria, a process important for quality control. The aims of this study were to evaluate (i) the effect of I/R on mitochondrial oxidative phosphorylation and its temporal relationship with mitophagy in hearts from obese rats and their age-matched controls, and (ii) the role of oxidative stress in these processes using melatonin, a free radical scavenger. METHODS: Male Wistar rats were divided into 4 groups: control (normal diet ± melatonin) and high-fat sucrose diet (HFSD ± melatonin). Rats received melatonin orally (10 mg/kg/day). After 16 weeks, hearts were removed and subjected to 40-min stabilization, and 25-min global ischaemia/10-min reperfusion for preparation of mitochondria. Mitochondrial oxidative phosphorylation was measured polarographically. Western blotting was used for evaluation of PINK1, Parkin, p62/SQSTM1 (p62) and TOM 70. Infarct size was measured using tetrazolium staining. RESULTS: Ischaemia and reperfusion respectively reduced and increased mitochondrial QO2 (state 3) and the ox-phos rate in both control and HFSD mitochondria, showing no major changes between the groups, while melatonin pretreatment had little effect. p62 as indicator of mitophagic flux showed up- and downregulation of mitophagy by ischaemia and reperfusion respectively, with melatonin having no significant effect. Melatonin treatment caused a significant reduction in infarct size in hearts from both control and diet groups. CONCLUSIONS: The results suggest that I/R (i) affects mitochondria from control and HFSD hearts similarly and (ii) melatonin-induced cardioprotection is not associated with reversal of mitochondrial dysfunction or changes in the PINK1/Parkin pathway.

The Combination Effect of Aspalathin and Phenylpyruvic Acid-2-O-ß-D-glucoside from Rooibos against Hyperglycemia-Induced Cardiac Damage: An In Vitro Study.

Recent evidence shows that rooibos compounds, aspalathin and phenylpyruvic acid-2-O-ß-D-glucoside (PPAG), can independently protect cardiomyocytes from hyperglycemia-related reactive oxygen species (ROS). While aspalathin shows more potency by enhancing intracellular antioxidant defenses, PPAG acts more as an anti-apoptotic agent. Thus, to further understand the protective capabilities of these compounds against hyperglycemia-induced cardiac damage, their combinatory effect was investigated and compared to metformin. An in vitro model of H9c2 cardiomyocytes exposed to chronic glucose concentrations was employed to study the impact of such compounds on hyperglycemia-induced damage. Here, high glucose exposure impaired myocardial substrate utilization by abnormally enhancing free fatty acid oxidation while concomitantly suppressing glucose oxidation. This was paralleled by altered expression of genes involved in energy metabolism including acetyl-CoA carboxylase (ACC), 5' AMP-activated protein kinase (AMPK), and peroxisome proliferator-activated receptor-alpha (PPARα). The combination treatment improved myocardial substrate metabolism, maintained mitochondrial membrane potential, and attenuated various markers for oxidative stress including nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity and glutathione content. It also showed a much-improved effect by ameliorating DNA damage when compared to metformin. The current study demonstrates that rooibos compounds offer unique cardioprotective properties against hyperglycemia-induced and potentially against diabetes-induced cardiac damage. These data also support further exploration of rooibos compounds to better assess the cardioprotective effects of different bioactive compound combinations.

An In Vivo/Ex Vivo Study Design to Investigate Effects of Chronic Conditions and Therapeutic Compounds on Adipose Stem Cells in Animal Models.

With the dramatic rise in the global prevalence of obesity and lack of success at addressing this public health issue, there is an urgency to develop new tools with which to study obesity and putative weight-loss products. Pre-adipocyte cell lines have been widely used as a model for adipocyte biology and obesity over the past four decades, but the applicability of results from these cell lines is limited. This chapter will describe an in vivo/ex vivo study design that can be employed to examine the effects of diets and other chronic physiological or pathophysiological conditions on the biology of adipose stem cells (ASCs), as a model for the progression and management of obesity. This type of study design is superior to short-term in vitro experiments in pre-adipocyte cell lines or ASCs, as chronic in vivo conditions cannot be recapitulated in cell culture. Rather, this in vivo/ex vivo study design provides researchers the opportunity to assess the progressive effects of long-term insults or interventions on the reprogramming of ASC behavior. In addition, this model allows us to study the metabolic effects of chronic conditions and therapeutic compounds at a systemic level as well as at the level of adipose tissue and ASCs, in order to provide a whole-body context for the findings.

Mitochondrial oxidative phosphorylation and mitophagy in myocardial ischaemia/reperfusion: effects of chloroquine.

AIM: The aim of this study was to evaluate the temporal relationship between mitochondrial oxidative phosphorylation and mitophagy in rat hearts subjected to ischaemia/reperfusion. Measurements were made at specific points during the experimental protocol (snapshot approach) and by assessments of mitophagic flux, using chloroquine pre-treatment. METHODS: Isolated working rat hearts were subjected to 25 or 30 minutes of global ischaemia/10 minutes of reperfusion. Half of each group received chloroquine (10 mg/kg, intraperitoneally) one hour before experimentation. Mitochondria were isolated after stabilisation, ischaemia and reperfusion, and oxidative phosphorylation was measured polarographically. Mitochondrial mitophagy markers were detected by Western blot analysis. RESULTS: Mitochondrial oxygen uptake (state 3) and oxidative phosphorylation rate were reduced by ischaemia and increased by reperfusion. Chloroquine pre-treatment increased both parameters. Using a snapshot approach, exposure to ischaemia ± reperfusion had little effect on mitochondrial PINK1, Parkin and p62/SQSTM1 expression. Ischaemia reduced Rab9 expression, and reperfusion upregulated the phosphor DRP1, phosphor/total DRP1 ratio and Rab9 levels. Chloroquine significantly reduced PINK1, p62/SQSTM1, Rab9 and particularly Parkin expression during reperfusion, without an effect on mitochondrial total and phospho DRP1 levels. CONCLUSIONS: Ischaemia/reperfusion-induced changes in mitochondrial oxidative phosphorylation function occurred concomitantly with changes in mitophagic flux. Pre-treatment with chloroquine profoundly affected mitochondrial function as well as the pattern of mitophagy during ischaemia/reperfusion.

Ataxia-Telangiectasia Mutated is located in cardiac mitochondria and impacts oxidative phosphorylation.

The absence of Ataxia-Telangiectasia mutated protein kinase (ATM) is associated with neurological, metabolic and cardiovascular defects. The protein has been associated with mitochondria and its absence results in mitochondrial dysfunction. Furthermore, it can be activated in the cytosol by mitochondrial oxidative stress and mediates a cellular anti-oxidant response through the pentose phosphate pathway (PPP). However, the precise location and function of ATM within mitochondria and its role in oxidative phosphorylation is still unknown. We show that ATM is found endogenously within cardiac myocyte mitochondria under normoxic conditions and is consistently associated with the inner mitochondrial membrane. Acute ex vivo inhibition of ATM protein kinase significantly decreased mitochondrial electron transfer chain complex I-mediated oxidative phosphorylation rate but did not decrease coupling efficiency or oxygen consumption rate during ß-oxidation. Chemical inhibition of ATM in rat cardiomyoblast cells (H9c2) significantly decreased the excited-state autofluorescence lifetime of enzyme-bound reduced NADH and its phosphorylated form, NADPH (NAD(P)H; 2.77 ± 0.26 ns compared to 2.57 ± 0.14 ns in KU60019-treated cells). This suggests an interaction between ATM and the electron transfer chain in the mitochondria, and hence may have an important role in oxidative phosphorylation in terminally differentiated cells such as cardiomyocytes.

Revisiting the Cardiotoxic Effect of Chloroquine.

PURPOSE: Cardiotoxicity is a well-known side effect of chloroquine. Several studies have proposed chloroquine as a potential anti-diabetic treatment but do not address this problem. The current study investigated the effect of ex vivo chloroquine treatment on (1) heart function and glucose uptake, (2) mitochondrial function and (3) in vivo treatment on heart function. METHODS: Control or obese male Wistar rats were used throughout. Dose responses of increasing chloroquine concentrations versus vehicle on cardiac function were measured using isolated, Langendorff-perfused hearts whilst glucose uptake and cell viability were determined in ventricular cardiomyocytes. Mitochondrial function was assessed with a Clark-type oxygraph (Hansatech) after ex vivo perfusion with 30 µM chloroquine versus vehicle. Animals were treated orally with 5 mg/kg/day chloroquine for 6 weeks. RESULTS: Acute chloroquine treatment of 10 µM was sufficient to significantly decrease heart function (p < 0.05) whilst 30 µM significantly reduced heart rate (p < 0.05). Chloroquine became toxic to isolated cardiomyocytes at high concentrations (100 µM), and had no effect on cardiomyocyte glucose uptake. Ex vivo treatment did not affect mitochondrial function, but chronic low-dose in vivo chloroquine treatment significantly decreased aortic output and total work in hearts (p < 0.005). CONCLUSION: Low and intermediate chloroquine doses administered either chronically or acutely are sufficient to result in myocardial dysfunction.

Melatonin and cardioprotection against ischaemia/reperfusion injury: What's new? A review.

Melatonin is a pleiotropic hormone with several functions. It binds to specific receptors and to a number of cytosolic proteins, activating a vast array of signalling pathways. Its potential to protect the heart against ischaemia/reperfusion damage has attracted much attention, particularly in view of its possible clinical applications. This review will focus mainly on the possible signalling pathways involved in melatonin-induced cardioprotection. In particular, the role of the melatonin receptors and events downstream of receptor activation, for example, the reperfusion injury salvage kinase (RISK), survivor activating factor enhancement (SAFE) and Notch pathways, the sirtuins, nuclear factor E2-related factor 2 (Nrf2) and translocases in the outer membrane (TOM70) will be discussed. Particular attention is given to the role of the mitochondrion in melatonin-induced cardioprotection. In addition, a brief overview will be given regarding the status quo of the clinical application of melatonin in humans.

Autophagy is essential for the maintenance of amino acids and ATP levels during acute amino acid starvation in MDAMB231 cells.

Autophagy plays a major role in the adaptive metabolic response of cancer cells during adverse conditions such as nutrient deprivation. However, specific data that assess metabolite profiles in context with adenosine triphosphate (ATP) availability and cell death susceptibility remain limited. Human breast cancer cells, MDAMB231, and normal breast epithelial cells, MCF12A, were subjected to short-term amino acid starvation and the cellular apoptotic and autophagic responses assessed. The role of autophagy in the control of cellular amino acid, ATP, free fatty acid, and glucose levels during amino acid starvation were compared. We demonstrate that breast cancer cells have an increased metabolic demand contributing to significant amino acid and ATP depletion in a nutrient-poor environment. Upregulation of autophagy was important for the generation of amino acids and free fatty acids and maintenance of cellular ATP levels. In contrast to normal cells, breast cancer cells were unable to maintain the response after 12 hours of amino acid starvation. Regulation of autophagic activity in these environments had indirect consequences on cell death susceptibility. Overall, our data provide support for autophagy as an important survival mechanism capable of providing metabolic substrates when cancer cells are faced with nutrient-deprived environments. SIGNIFICANCE OF STUDY: The results obtained in this study helps to expand our current knowledge on how cells respond to environmental changes; the biochemical and metabolic consequences and the physiological processes activated in response. The environmental stress applied in this study is relevant to tumour physiology, and results can be translated to cancer therapeutic and clinical research areas, ultimately assisting in the specific targeting of cancer cells while avoiding harm to normal cells.

Myocardial Glucose Clearance by Aspalathin Treatment in Young, Mature, and Obese Insulin-Resistant Rats.

Rooibos, an indigenous South African plant ingested as herbal tea, is well known for its antioxidant effects. This in vitro study investigated aspalathin (C21H24O11), a dihydrochalcone unique to rooibos, for hypoglycemic effects in the context of age- and obesity-induced insulin resistance and the mechanisms involved. Male Wistar rats were allocated into three groups: 16 - 30 weeks feeding with either standard rat chow or a high-caloric diet, or 6 - 10 weeks feeding with standard rat chow. Ventricular cardiomyocytes were isolated by collagenase perfusion digestion, and glucose uptake was determined by 2-[3H]-deoxyglucose accumulation. Viability was tested by trypan blue exclusion or propidium iodide staining. The high-caloric diet significantly increased body weight gain (508.5 ± 50.0 vs. 417.3 ± 40.0 g), visceral adiposity (42.30 ± 10.1 vs. 21.75 ± 7.0 g), and fasting blood glucose (5.7 ± 0.4 vs. 4.7 ± 0.1 mM). Aspalathin (10 µM for 90 min) induced 2-[3H]-deoxyglucose uptake in young cardiomyocytes (37.2 ± 13.9 vs. 25.7 ± 2.5 pmol 2-[3H]-deoxyglucose/mg protein) and enhanced insulin-mediated 2-[3H]-deoxyglucose uptake in control cells (32.4 ± 6.4 vs. 23.5 ± 10.0 pmol 2-[3H]-deoxyglucose/mg protein), but failed to induce 2-[3H]-deoxyglucose uptake in high-caloric diet cells. Aspalathin induced glucose uptake in insulin-sensitive cardiomyocytes from young and aged rats, but not in high-caloric diet animals and enhanced the actions of insulin through a PI3K-dependent mechanism, resulting in an additive response.

Polyphenol-Enriched Fractions of Cyclopia intermedia Selectively Affect Lipogenesis and Lipolysis in 3T3-L1 Adipocytes.

Cyclopia species are increasingly investigated as sources of phenolic compounds with potential as therapeutic agents. Recently, we demonstrated that a crude polyphenol-enriched organic fraction (CPEF) of Cyclopia intermedia, currently forming the bulk of commercial production, decreased lipid content in 3T3-L1 adipocytes and inhibited body weight gain in obese db/db mice. The aim of the present study was to determine whether a more effective product and/or one with higher specificity could be obtained by fractionation of the CPEF by purposely increasing xanthone and benzophenone levels. Fractionation of the CPEF using high performance counter-current chromatography (HPCCC) resulted in four fractions (F1-F4), predominantly containing iriflophenone-3-C-ß-D-glucoside-4-O-ß-D-glucoside (benzophenone: F1), hesperidin (flavanone: F2), mangiferin (xanthone: F3), and neoponcirin (flavone: F4), as quantified by high-performance liquid chromatography with diode array detection (HPLC-DAD), and confirmed by LC-DAD with mass spectrometric (MS) and tandem MS (MSE) detection. All fractions inhibited lipid accumulation in 3T3-L1 pre-adipocytes and decreased lipid content in mature 3T3-L1 adipocytes, although their effects were concentration-dependent. F1-F3 stimulated lipolysis in mature adipocytes. Treatment of mature adipocytes with F1 and F2 increased the messenger RNA expression of hormone sensitive lipase, while treatment with F1 and F4 increased uncoupling protein 3 expression. In conclusion, HPCCC resulted in fractions with different phenolic compounds and varying anti-obesity effects. The activities of fractions were lower than the CPEF; thus, fractionation did not enhance activity within a single fraction worthwhile for exploitation as a nutraceutical product, which illustrates the importance of considering synergistic effects in plant extracts.

Role of melatonin in glucose uptake by cardiomyocytes from insulin-resistant Wistar rats.

AIM: Melatonin supplementation reduces insulin resistance and protects the heart in obese rats. However, its role in myocardial glucose uptake remains unknown. This study investigated the effect of short-term melatonin treatment on glucose uptake by cardiomyocytes isolated from obese and insulin-resistant rats. METHODS: Cardiomyocytes were isolated from obese rats fed a high-calorie diet for 16 to 23 weeks, their age-matched controls, as well as young control rats aged four to eight weeks. After incubation with melatonin with or without insulin, glucose uptake was initiated by the addition of 2-deoxy-D- [3H] glucose and measured after 30 minutes. Additional control and obese rats received melatonin in the drinking water (4 mg/kg/day) for the last six weeks of feeding (20 weeks) and glucose uptake was determined in isolated cardiomyocytes after incubation with insulin. Intraperitoneal glucose tolerance and biometric parameters were also measured. RESULTS: Obese rats (fed for more than 20 weeks) developed glucose intolerance. Cardiomyocytes isolated from these obese rats had a reduced response to insulin-stimulated glucose uptake (ISGU) (p < 0.05). Melatonin administration in vitro had no effect on glucose uptake per se. However, it increased ISGU by cardiomyocytes from the young rats (p < 0.05), while having no effect on ISGU by cardiomyocytes from the older control and obese groups. Melatonin in vivo had no significant effect on glucose tolerance, but it increased basal (p < 0.05) and ISGU by cardiomyocytes from the obese rats (50.1 ± 1.7 vs 32.1 ± 5.1 pmol/mg protein/30 min, p < 0.01). CONCLUSION: These data suggest that short-term melatonin treatment in vivo but not in vitro improved glucose uptake and insulin responsiveness of cardiomyocytes in obesity and insulin-resistance states.

The Role of MKP-1 in Insulin-Induced Cardioprotection.

PURPOSE: The mitogen-activated protein kinase phosphatases (MKPs) are a family of dual-specificity phosphatases that inactivate MAPKs by dephosphorylation. Impairment of MKP-1 expression in insulin resistance has been suggested to affect the cardioprotective properties of insulin. We hypothesized that manipulation of its activity during myocardial ischaemia/reperfusion of control as well as insulin-resistant rats may affect the outcome. METHODS: Hearts from 16 week dietary induced obese Wistar rats and their age matched controls were isolated, perfused in the working mode and subjected to 15 min global ischaemia / 30 min reperfusion or 35 min coronary artery ligation/ 60 min reperfusion. Hearts received insulin (1mIU/ml), a MKP-1 inhibitor (sanguinarine 2.5uM), or insulin + sanguinarine for 15 min pre- and 10 min post-ischaemia. Endpoints were functional recovery and infarct size. Hearts from control and experimental groups were freeze-clamped either immediately after removal from the animal (baseline) or at 10 min reperfusion after global ischaemia and Western blot analysis done for total and phosphorylated MKP-1. RESULTS: Insulin treatment significantly increased total work recovery while sanguinarine abolished the insulin-mediated protection. Insulin had no effect on infarct size while sanguinarine reduced infarct size. Insulin increased while sanguinarine attenuated phosphorylation of MKP-1 at 10 min reperfusion. CONCLUSION: Inhibition of MKP-1 with sanguinarine abolished the insulin-induced improvement in functional recovery, but reduced infarct size. Although the data suggest a role for this phosphatase in insulin-induced cardioprotection, the multiple downstream effects of insulin hamper interpretation of the data obtained. In addition, the effects of sanguinarine per se in myocardial ischaemia/reperfusion need to be further elucidated.

The Transcription Profile Unveils the Cardioprotective Effect of Aspalathin against Lipid Toxicity in an In Vitro H9c2 Model.

Aspalathin, a C-glucosyl dihydrochalcone, has previously been shown to protect cardiomyocytes against hyperglycemia-induced shifts in substrate preference and subsequent apoptosis. However, the precise gene regulatory network remains to be elucidated. To unravel the mechanism and provide insight into this supposition, the direct effect of aspalathin in an isolated cell-based system, without the influence of any variables, was tested using an H9c2 cardiomyocyte model. Cardiomyocytes were exposed to high glucose (33 mM) for 48 h before post-treatment with or without aspalathin. Thereafter, RNA was extracted and RT2 PCR Profiler Arrays were used to profile the expression of 336 genes. Results showed that, 57 genes were differentially regulated in the high glucose or high glucose and aspalathin treated groups. Search Tool for the Retrieval of Interacting Genes/Proteins (STRING) analysis revealed lipid metabolism and molecular transport as the biological processes altered after high glucose treatment, followed by inflammation and apoptosis. Aspalathin was able to modulate key regulators associated with lipid metabolism (Adipoq, Apob, CD36, Cpt1, Pparγ, Srebf1/2, Scd1 and Vldlr), insulin resistance (Igf1, Akt1, Pde3 and Map2k1), inflammation (Il3, Il6, Jak2, Lepr, Socs3, and Tnf13) and apoptosis (Bcl2 and Chuk). Collectively, our results suggest that aspalathin could reverse metabolic abnormalities by activating Adipoq while modulating the expression of Pparγ and Srebf1/2, decreasing inflammation via Il6/Jak2 pathway, which together with an observed increased expression of Bcl2 prevents myocardium apoptosis.