RESUMO
BACKGROUND: Leukoreduced whole blood (LR-WB) has received renewed attention as alternative to component-based transfusion in trauma. According to the manufacturer's instructions, leukoreduction should be carried out within 8 h after collection. This study assessed impact of (1) WB collection bag, (2) LR filtration, and (3) timing of filtration on in vitro quality. STUDY DESIGN AND METHODS: WB collected into different vendor bags was held at room temperature for <8 h or >16 h but <24 h prior to LR. In vitro quality was assessed before and after filtration, and throughout 3 weeks of storage at 4°C. Cell count and hemoglobin levels were determined by hematology analyzer, platelet activation, and responsiveness to ADP by surface expression of P-selectin by flow cytometry, hemolysis by HemoCue, and metabolic parameters by blood gas analyzer. Hemostatic properties were assessed by rotational thromboelastometry. Plasma protein activities and clotting times were determined by automated coagulation analyzer or quantitative immunoblotting. RESULTS: Bag type had no impact on WB in vitro quality. LR by filtration had some impact, but is aligned with data in the literature. The time between donation and filtration resulted in some statistically significant differences in metabolic activity, platelet yield, platelet activation, and factor protein activity initially; however, these differences in in vitro quality attributes decreased throughout 21-day cold storage. CONCLUSION: WB hold time showed only a minor impact on WB in vitro quality, so it may be possible for blood processing facilities to explore extended hold times prior to filtration in order to provide greater operational flexibility.
Assuntos
Preservação de Sangue/métodos , Contagem de Células Sanguíneas , Temperatura Baixa , Hemólise , Hemostasia , Humanos , Procedimentos de Redução de Leucócitos/métodos , Ativação Plaquetária , TromboelastografiaRESUMO
Metabolic disturbances and oxidative stress have been highlighted as potential causative factors for the development of diabetic cardiomyopathy. The ß-blocker metoprolol is known to improve function in the diabetic rat heart and ameliorates the sequelae associated with oxidative stress, without lowering oxidative stress. The antioxidant ascorbic acid is known to improve function in the diabetic rat heart. We tested whether a combination of ascorbic acid and metoprolol treatment would improve function further than each drug individually. Control and streptozotocin-induced diabetic Wistar rats were treated with metoprolol (15 mg·(kg body mass)(-1)·day(-1), via an osmotic pump) and (or) ascorbic acid (1000 mg·(kg body mass)(-1)·day(-1), via their drinking water). To study the effect of treatment on the development of dysfunction, we examined time points before (5 weeks diabetic) and after (7 weeks diabetic) development of overt systolic dysfunction. Echocardiography and working-heart-perfusion were used to assess cardiac function. Blood and tissue samples were collected to assess the severity of disease and oxidative stress. While both drugs improved function, only ascorbic acid had effects on oxidative damage. Combination treatment had a more pronounced improvement in function. Our ß-blocker + antioxidant treatment strategy focused on oxidative stress, not diabetes specifically; therefore, it may prove useful in other diseases where oxidative stress contributes to the pathology.
Assuntos
Antagonistas de Receptores Adrenérgicos beta 1/uso terapêutico , Antioxidantes/uso terapêutico , Ácido Ascórbico/uso terapêutico , Cardiomiopatias Diabéticas/prevenção & controle , Metoprolol/uso terapêutico , Miocárdio/patologia , Animais , Cardiomiopatias Diabéticas/patologia , Cardiomiopatias Diabéticas/fisiopatologia , Quimioterapia Combinada , Coração/efeitos dos fármacos , Coração/fisiopatologia , Masculino , Estresse Oxidativo , Ratos WistarRESUMO
Increased awareness of obesity has led to a dietary shift toward "heart-friendly" vegetable oils containing omega-6 polyunsaturated fatty acid (omega-6 PUFA). In addition to its beneficial effects, omega-6 PUFA also exhibits proinflammatory and prooxidative properties. We hypothesized that chronic dietary omega-6 PUFA can induce free radical generation, predisposing the cardiac mitochondria to oxidative damage. Male Wistar rats were fed a diet supplemented with 20% w/w sunflower oil, rich in omega-6 PUFA (HP) or normal laboratory chow (LP) for 4 weeks. HP feeding augmented phospholipase A(2) activity and breakdown of cardiolipin, a mitochondrial phospholipid. HP hearts also demonstrated elevated inducible nitric oxide synthase expression, loss of Mn superoxide dismutase, and increased mitochondrial nitrotyrosine levels. In these hearts, oxidative damage to mitochondrial DNA (mDNA) was demonstrated by 8-hydroxyguanosine immunopositivity, overexpression of DNA repair enzymes, and a decrease in the mRNA expression of specific respiratory subunits encoded by the mDNA. Functionally, at higher workloads, HP hearts also demonstrated a greater decline in cardiac work than LP, suggesting a compromised mitochondrial reserve. Our study, for the first time, demonstrates that consumption of a high fat diet rich in omega-6 PUFA for only 4 weeks instigates mitochondrial nitrosative damage and causes cardiac dysfunction at high afterloads.
Assuntos
Dieta , Ácidos Graxos Ômega-6/metabolismo , Cardiopatias/etiologia , Mitocôndrias Cardíacas/metabolismo , Tirosina/análogos & derivados , Animais , Western Blotting , DNA Mitocondrial/genética , DNA Mitocondrial/metabolismo , Imunofluorescência , Expressão Gênica/efeitos dos fármacos , Cardiopatias/metabolismo , Peróxidos Lipídicos/metabolismo , Masculino , Óxido Nítrico Sintase Tipo II/metabolismo , Estresse Oxidativo , Fosfolipases A/metabolismo , Óleos de Plantas/química , Ratos , Ratos Wistar , Óleo de Girassol , Superóxido Dismutase/metabolismo , Tirosina/metabolismoRESUMO
BACKGROUND: Gender influences the cardiac response to prolonged increases in workload, with differences at structural, functional, and molecular levels. However, it is unknown if post-ischemic function or metabolism of female hypertrophied hearts differ from male hypertrophied hearts. Thus, we tested the hypothesis that gender influences post-ischemic function of pressure-overload hypertrophied hearts and determined if the effect of gender on post-ischemic outcome could be explained by differences in metabolism, especially the catabolic fate of glucose. METHODS: Function and metabolism of isolated working hearts from sham-operated and aortic-constricted male and female Sprague-Dawley rats before and after 20 min of no-flow ischemia (N = 17 to 27 per group) were compared. Parallel series of hearts were perfused with Krebs-Henseleit solution containing 5.5 mM [5-3H/U-14C]-glucose, 1.2 mM [1-14C]-palmitate, 0.5 mM [U-14C]-lactate, and 100 mU/L insulin to measure glycolysis and glucose oxidation in one series and oxidation of palmitate and lactate in the second. Statistical analysis was performed using two-way analysis of variance. The sequential rejective Bonferroni procedure was used to correct for multiple comparisons and tests. RESULTS: Female gender negatively influenced post-ischemic function of non-hypertrophied hearts, but did not significantly influence function of hypertrophied hearts after ischemia such that mass-corrected hypertrophied heart function did not differ between genders. Before ischemia, glycolysis was accelerated in hypertrophied hearts, but to a greater extent in males, and did not differ between male and female non-hypertrophied hearts. Glycolysis fell in all groups after ischemia, except in non-hypertrophied female hearts, with the reduction in glycolysis after ischemia being greatest in males. Post-ischemic glycolytic rates were, therefore, similarly accelerated in hypertrophied male and female hearts and higher in female than male non-hypertrophied hearts. Glucose oxidation was lower in female than male hearts and was unaffected by hypertrophy or ischemia. Consequently, non-oxidative catabolism of glucose after ischemia was lowest in male non-hypertrophied hearts and comparably elevated in hypertrophied hearts of both sexes. These differences in non-oxidative glucose catabolism were inversely related to post-ischemic functional recovery. CONCLUSION: Gender does not significantly influence post-ischemic function of hypertrophied hearts, even though female sex is detrimental to post-ischemic function in non-hypertrophied hearts. Differences in glucose catabolism may contribute to hypertrophy-induced and gender-related differences in post-ischemic function.
Assuntos
Cardiomegalia/fisiopatologia , Coração/fisiopatologia , Isquemia Miocárdica/fisiopatologia , Animais , Feminino , Glucose/metabolismo , Glicólise , Masculino , Oxirredução , Ratos , Ratos Sprague-Dawley , Fatores SexuaisRESUMO
OBJECTIVE: Coupling of glucose oxidation to glycolysis is lower in hypertrophied than in non-hypertrophied hearts, contributing to the compromised mechanical performance of hypertrophied hearts. Here, we describe studies to test the hypothesis that low coupling of glucose oxidation to glycolysis in hypertrophied hearts is due to reduced activity and/or expression of the pyruvate dehydrogenase complex (PDC). METHODS: We examined the effects of dichloroacetate (DCA), an inhibitor of PDC kinase, and of alterations in exogenous palmitate supply on coupling of glucose oxidation to glycolysis in isolated working hypertrophied and control hearts from aortic-constricted and sham-operated male Sprague-Dawley rats. It was anticipated that the addition of DCA or the absence of palmitate would promote PDC activation and consequently normalize coupling between glycolysis and glucose oxidation in hypertrophied hearts if our hypothesis was correct. RESULTS: Addition of DCA or removal of palmitate improved coupling of glucose oxidation to glycolysis in control and hypertrophied hearts. However, coupling remained substantially lower in hypertrophied hearts. PDC activity in extracts of hypertrophied hearts was similar to or higher than in extracts of control hearts under all perfusion conditions. No differences were observed between hypertrophied and control hearts with respect to expression of PDC, PDC kinase, or PDC phosphatase. CONCLUSIONS: Low coupling of glucose oxidation to glycolysis in hypertrophied hearts is not due to a reduction in PDC activity or subunit expression indicating that other mechanism(s) are responsible.
Assuntos
Glucose/metabolismo , Hipertrofia Ventricular Esquerda/metabolismo , Complexo Piruvato Desidrogenase/fisiologia , Animais , Expressão Gênica , Glicólise , Hipertrofia Ventricular Esquerda/enzimologia , Masculino , Oxirredução , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Piruvato Desidrogenase (Lipoamida)-Fosfatase/metabolismo , Piruvato Desidrogenase Quinase de Transferência de Acetil , Complexo Piruvato Desidrogenase/genética , Complexo Piruvato Desidrogenase/metabolismo , RNA Mensageiro/genética , Ratos , Ratos Sprague-DawleyRESUMO
Leptin has previously been shown to stimulate fatty acid oxidation independent of AMP-activated protein kinase (AMPK). Nitric oxide and p38 mitogen activated protein kinase (MAPK) are known effectors of leptin signaling. The aim of the present study was to determine whether nitric oxide and p38 MAPK mediate the stimulation of leptin by MAPK. Hearts from male Sprague-Dawley rats were mounted on the isolated perfused working heart in the presence or absence of leptin (1.9 nM), N-Nitro-L-Arginine Methyl Ester (L-NAME) (3 microM), the specific p38 MAPK inhibitor 4-[4-(4-Fluorophenyl)-5-(4-pyridinyl)-1H-imidazol-2-yl] phenol (SB202190, 2 microM) and the specific STAT-3 inhibitor (E)-2-Cyano-3-(3,4-dihydrophenyl)-N-(phenylmethyl)-2-propenamide (AG490, 5 microM) for the measurement of substrate metabolism and function. AMPK and carnitine palimitoyltransferase-1 activity, nitrate/nitrite levels, STAT-3 phosphorylation and p38 MAPK phosphorylation were measured. To assess mitochondrial function, hearts were perfused with or without leptin prior to the isolation of mitochondria. Leptin stimulated fatty acid oxidation and decreased cardiac function, associated with the activation of STAT-3 and p38 MAPK and an increase in tissue nitrate/nitrite levels; the effect on function was ameliorated and the effect on fatty acid oxidation was prevented by L-NAME, B202190 and AG490. L-NAME lowered tissue nitrate/nitrite levels, and prevented the phosphorylation of p38, whereas SB202190 had no effect on tissue nitrate/nitrite levels. AG490 also lowered tissue nitrate/nitrite levels. Leptin had no effect on fatty acid-dependent mitochondrial respiration or uncoupling activity, but, surprisingly, stimulated pyruvate-dependent mitochondrial respiration. These data indicate that leptin stimulates fatty acid oxidation by a STAT-3-nitric oxide-p38 MAPK-dependent mechanism. The target of the pathway is upstream of the mitochondria.
Assuntos
Ácidos Graxos/metabolismo , Coração/efeitos dos fármacos , Leptina/farmacologia , Miocárdio/metabolismo , Óxido Nítrico/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Glucose/metabolismo , Técnicas In Vitro , Ácido Láctico/biossíntese , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Nitratos/metabolismo , Nitritos/metabolismo , Oxirredução/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Fator de Transcrição STAT3/metabolismoRESUMO
Substrate use switches from fatty acids toward glucose in pressure overload-induced cardiac hypertrophy with an acceleration of glycolysis being characteristic. The activation of AMP-activated protein kinase (AMPK) observed in hypertrophied hearts provides one potential mechanism for the acceleration of glycolysis. Here, we directly tested the hypothesis that AMPK causes the acceleration of glycolysis in hypertrophied heart muscle cells. The H9c2 cell line, derived from the embryonic rat heart, was treated with arginine vasopressin (AVP; 1 microM) to induce a cellular model of hypertrophy. Rates of glycolysis and oxidation of glucose and palmitate were measured in nonhypertrophied and hypertrophied H9c2 cells, and the effects of inhibition of AMPK were determined. AMPK activity was inhibited by 6-[4-(2-piperidin-1- yl-ethoxy)-phenyl]-3-pyridin-4-yl-pyrrazolo-[1,5-a]pyrimidine (compound C) or by adenovirus-mediated transfer of dominant negative AMPK. Compared with nonhypertrophied cells, glycolysis was accelerated and palmitate oxidation was reduced with no significant alteration in glucose oxidation in hypertrophied cells, a metabolic profile similar to that of intact hypertrophied hearts. Inhibition of AMPK resulted in the partial reduction of glycolysis in AVP-treated hypertrophied H9c2 cells. Acute exposure of H9c2 cells to AVP also activated AMPK and accelerated glycolysis. These elevated rates of glycolysis were not altered by AMPK inhibition but were blocked by agents that interfere with Ca(2+) signaling, including extracellular EGTA, dantrolene, and 2-aminoethoxydiphenyl borate. We conclude that the acceleration of glycolysis in AVP-treated hypertrophied heart muscle cells is partially dependent on AMPK, whereas the acute glycolytic effects of AVP are AMPK independent and at least partially Ca(2+) dependent.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Arginina Vasopressina/metabolismo , Cardiomegalia/metabolismo , Miócitos Cardíacos/enzimologia , Vasoconstritores/metabolismo , Proteínas Quinases Ativadas por AMP/antagonistas & inibidores , Animais , Arginina Vasopressina/farmacologia , Comunicação Autócrina/efeitos dos fármacos , Comunicação Autócrina/fisiologia , Cálcio/metabolismo , Cardiomegalia/patologia , Linhagem Celular , Metabolismo Energético/efeitos dos fármacos , Metabolismo Energético/fisiologia , Glucose/metabolismo , Glicólise/efeitos dos fármacos , Glicólise/fisiologia , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Comunicação Parácrina/efeitos dos fármacos , Comunicação Parácrina/fisiologia , Pirazóis/farmacologia , Pirimidinas/farmacologia , Ratos , Vasoconstritores/farmacologiaRESUMO
The effects of diabetes on heart function may be initiated or compounded by the exaggerated reliance of the diabetic heart on fatty acids and ketones as metabolic fuels. beta-Blocking agents such as metoprolol have been proposed to inhibit fatty acid oxidation. We hypothesized that metoprolol would improve cardiac function by inhibiting fatty acid oxidation and promoting a compensatory increase in glucose utilization. We measured ex vivo cardiac function and substrate utilization after chronic metoprolol treatment and acute metoprolol perfusion. Chronic metoprolol treatment attenuated the development of cardiac dysfunction in streptozotocin (STZ)-diabetic rats. After chronic treatment with metoprolol, palmitate oxidation was increased in control hearts but decreased in diabetic hearts without affecting myocardial energetics. Acute treatment with metoprolol during heart perfusions led to reduced rates of palmitate oxidation, stimulation of glucose oxidation, and increased tissue ATP levels. Metoprolol lowered malonyl-CoA levels in control hearts only, but no changes in acetyl-CoA carboxylase phosphorylation or AMP-activated protein kinase activity were observed. Both acute metoprolol perfusion and chronic in vivo metoprolol treatment led to decreased maximum activity and decreased sensitivity of carnitine palmitoyltransferase I to malonyl-CoA. Metoprolol also increased sarco(endo)plasmic reticulum Ca(2+)-ATPase expression and prevented the reexpression of atrial natriuretic peptide in diabetic hearts. These data demonstrate that metoprolol ameliorates diabetic cardiomyopathy and inhibits fatty acid oxidation in streptozotocin-induced diabetes. Since malonyl-CoA levels are not increased, the reduction in total carnitine palmitoyltransferase I activity is the most likely factor to explain the decrease in fatty acid oxidation. The metabolism changes occur in parallel with changes in gene expression.
Assuntos
Antagonistas Adrenérgicos beta/farmacologia , Diabetes Mellitus Experimental/tratamento farmacológico , Metabolismo Energético/efeitos dos fármacos , Hemodinâmica/efeitos dos fármacos , Metoprolol/farmacologia , Miocárdio/metabolismo , Proteínas Quinases Ativadas por AMP , Acetil-CoA Carboxilase/metabolismo , Trifosfato de Adenosina/metabolismo , Antagonistas Adrenérgicos beta/uso terapêutico , Animais , Fator Natriurético Atrial/metabolismo , Débito Cardíaco/efeitos dos fármacos , Carnitina O-Palmitoiltransferase/antagonistas & inibidores , Carnitina O-Palmitoiltransferase/metabolismo , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Glucose/metabolismo , Frequência Cardíaca/efeitos dos fármacos , Masculino , Malonil Coenzima A/metabolismo , Metoprolol/uso terapêutico , Complexos Multienzimáticos/metabolismo , Miocárdio/enzimologia , Oxirredução , Ácido Palmítico/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Complexo Piruvato Desidrogenase/metabolismo , Ratos , Ratos Wistar , Retículo Sarcoplasmático/metabolismo , ATPases Transportadoras de Cálcio do Retículo Sarcoplasmático/metabolismoRESUMO
The metabolic actions of the antidiabetic agent metformin reportedly occur via the activation of the AMP-activated protein kinase (AMPK) in the heart and other tissues in the presence or absence of changes in cellular energy status. In this study, we tested the hypothesis that metformin has AMPK-independent effects on metabolism in heart muscle. Fatty acid oxidation and glucose utilization (glycolysis and glucose uptake) were measured in isolated working hearts from halothane-anesthetized male Sprague-Dawley rats and in cultured heart-derived H9c2 cells in the absence or in the presence of metformin (2 mM). Fatty acid oxidation and glucose utilization were significantly altered by metformin in hearts and H9c2 cells. AMPK activity was not measurably altered by metformin in either model system, and no impairment of energetic state was observed in the intact hearts. Furthermore, the inhibition of AMPK by 6-[4-(2-piperidin-1-yl-ethoxy)-phenyl]-3-pyridin-4-yl-pyyrazolo[1,5-a] pyrimidine (Compound C), a well-recognized pharmacological inhibitor of AMPK, or the overexpression of a dominant-negative form of AMPK failed to prevent the metabolic actions of metformin in H9c2 cells. The exposure of H9c2 cells to inhibitors of p38 mitogen-activated protein kinase (p38 MAPK) or protein kinase C (PKC) partially or completely abrogated metformin-induced alterations in metabolism in these cells, respectively. Thus the metabolic actions of metformin in the heart muscle can occur independent of changes in AMPK activity and may be mediated by p38 MAPK- and PKC-dependent mechanisms.
Assuntos
Metabolismo Energético/efeitos dos fármacos , Coração/efeitos dos fármacos , Hipoglicemiantes/farmacologia , Metformina/farmacologia , Miocárdio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteínas Quinases Ativadas por AMP , Nucleotídeos de Adenina/metabolismo , Animais , Débito Cardíaco/efeitos dos fármacos , Linhagem Celular , Ácidos Graxos/metabolismo , Glucose/metabolismo , Glicogênio/metabolismo , Frequência Cardíaca/efeitos dos fármacos , Masculino , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/metabolismo , Miocárdio/enzimologia , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Oxirredução , Fosfocreatina/metabolismo , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Triglicerídeos/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
Accelerated glycolysis in hypertrophied hearts may be a compensatory response to reduced energy production from long-chain fatty acid oxidation with 5'-AMP-activated protein kinase (AMPK) functioning as a cellular signal. Therefore, we tested the hypothesis that enhanced fatty acid oxidation improves energy status and normalizes AMPK activity and glycolysis in hypertrophied hearts. Glycolysis, fatty acid oxidation, AMPK activity, and energy status were measured in isolated working hypertrophied and control hearts from aortic-constricted and sham-operated male Sprague-Dawley rats. Hearts from halothane (3-4%)-anesthetized rats were perfused with KH solution containing either palmitate, a long-chain fatty acid, or palmitate plus octanoate, a medium-chain fatty acid whose oxidation is not impaired in hypertrophied hearts. Compared with control, fatty acid oxidation was lower in hypertrophied hearts perfused with palmitate, whereas it increased to similar values in both groups with octanoate plus palmitate. Glycolysis was accelerated in palmitate-perfused hypertrophied hearts and was normalized in hypertrophied hearts by the addition of octanoate. AMPK activity was increased three- to sixfold with palmitate alone and was reduced to control values by octanoate plus palmitate. Myocardial energy status improved with the addition of octanoate but did not differ between groups. Our findings, particularly the correspondence between glycolysis and AMPK activity, provide support for the view that activation of AMPK is responsible, in part, for the acceleration of glycolysis in cardiac hypertrophy. Additionally, they indicate myocardial AMPK is activated by energy state-independent mechanisms in response to pressure overload, demonstrating AMPK is more than a sensor of the heart's energy status.
Assuntos
Metabolismo Energético , Ácidos Graxos/metabolismo , Coração/fisiopatologia , Hipertrofia Ventricular Esquerda/fisiopatologia , Complexos Multienzimáticos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases Ativadas por AMP , Adaptação Fisiológica , Animais , Pressão Sanguínea , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Flexibility in substrate selection is essential for the heart to maintain production of energy and contractile function, and is managed through multiple mechanisms including PPAR-alpha and AMP-activated protein kinase (AMPK). Rats injected with 55 mg/kg STZ (D55) were kept for 4 days (acute diabetes; D55-A) prior to termination. Fatty acid (FA) oxidation increased in D55-A hearts, with no significant change in gene expression of PPAR-alpha, or its downstream targets. However, both AMPK and ACC phosphorylation were significantly higher in these hearts, effects that were reversed by insulin. Unexpectedly, when the duration of diabetes in D55 rats was extended to 6 weeks (chronic diabetes; D55-C), AMPK and ACC phosphorylation were comparable in control and D55-C hearts. In D55-C rat hearts, lack of AMPK activation was closely associated to an overload of plasma and cardiac lipids. To validate the relationship between lipids and cardiac AMPK activation, we either induced more severe diabetes (100 mg/kg STZ to provoke both hyperglycemia and hyperlipidemia acutely; D100-A) or infused intralipid (IL) to enlarge circulating lipids. There was no difference in cardiac AMPK and ACC phosphorylation in D100-A rats compared to control. Measurement of AMPK and ACC phosphorylation in control and D55-A hearts revealed that their phosphorylation was inhibited by acute intralipid infusion. Our data suggest that activation of AMPK is an adaptation that would ensure adequate cardiac energy production when glucose utilization is compromised. However, in severe diabetes, with the addition of augmented plasma and heart lipids, AMPK activation is prevented, and control of FA oxidation is likely through alternate mechanisms. Given that AMPK plays an important role in preventing cardiac ischemic/reperfusion damage, it is possible that in these diabetic hearts, the accelerated damage observed during exposure to ischemia/reperfusion could be a likely outcome of a compromised activation of AMPK.
Assuntos
Diabetes Mellitus Experimental/enzimologia , Ácidos Graxos/metabolismo , Insulina/deficiência , Proteínas Quinases Ativadas por AMP , Animais , Ativação Enzimática , Hiperglicemia/induzido quimicamente , Hiperglicemia/enzimologia , Hiperlipidemias/induzido quimicamente , Hiperlipidemias/enzimologia , Masculino , Complexos Multienzimáticos , Traumatismo por Reperfusão Miocárdica/enzimologia , Traumatismo por Reperfusão Miocárdica/prevenção & controle , Oxirredução , PPAR alfa/metabolismo , Proteínas Serina-Treonina Quinases , Ratos , Ratos WistarRESUMO
Glucocorticoid therapy is often associated with impaired insulin sensitivity and cardiovascular disease. The present study was designed to evaluate cardiac fatty acid (FA) composition and metabolism following acute dexamethasone (Dex) treatment. Using the euglycemic hyperinsulinemic clamp, rats injected with Dex demonstrated a reduced glucose infusion rate. This whole body insulin resistance was also associated with a heart-specific increase in pyruvate dehydrogenase kinase 4 gene expression and a reduction in the rate of glucose oxidation. Dex treatment increased basal and postheparin plasma lipolytic activity. In the heart, palmitic and oleic acid levels were higher after 4 h of Dex and decreased to control (CON) levels within 8 h. Measurement of polyunsaturated FAs demonstrated a drop in linoleic and gamma-linolenic acid, with an increase in arachidonic acid (AA) after acute Dex injection. Tissue FA can be either oxidized or stored as triglyceride (TG). At 4 h, Dex augmented cardiac TG accumulation. However, this increase in tissue TG could not be maintained, such that at 8 h following Dex, TG declined to CON levels. AMP-activated protein kinase (AMPK) activation is known to promote FA oxidation through its control of acetyl-CoA carboxylase (ACC). Acute Dex promoted ACC phosphorylation, and increased cardiac palmitate oxidation, likely through its effects in increasing AMPK phosphorylation and total AMPK protein and gene expression. Whether these acute effects of Dex on FA oxidation, TG storage, and arachidonic acid accumulation can be translated into increased cardiovascular risk following chronic therapy has yet to be determined.
Assuntos
Dexametasona/administração & dosagem , Ácidos Graxos/metabolismo , Coração/efeitos dos fármacos , Resistência à Insulina/fisiologia , Miocárdio/metabolismo , Adaptação Fisiológica/efeitos dos fármacos , Adaptação Fisiológica/fisiologia , Animais , Masculino , Taxa de Depuração Metabólica , Ratos , Ratos Wistar , Distribuição TecidualRESUMO
The fraction of glucose passing through glycolysis that is oxidized is low in hypertrophied hearts, a pattern of glucose use associated with poor postischemic contractile function. We tested the hypothesis that trimetazidine, a partial 3-ketoacyl coenzyme A thiolase inhibitor, would stimulate glucose oxidation and, thereby, improve fractional glucose oxidation and postischemic function of hypertrophied hearts. Function, glycolysis, and oxidation of glucose, lactate, and palmitate were measured before and after global no-flow ischemia in isolated working hearts from sham-operated (control) and aortic-constricted (hypertrophied) male Sprague-Dawley rats in the presence or absence of 1 microM trimetazidine. Heart function was significantly improved by trimetazidine after ischemia, but only in hypertrophied hearts, with function improving to values in untreated control hearts. This effect occurred in association with relatively minor changes in oxidative metabolism. However, trimetazidine reduced glycolysis by approximately 30% but did so only in hypertrophied hearts, an unexpected novel action of this agent that resulted in a larger fractional oxidation of glucose, effectively normalizing it in hypertrophied hearts. Thus, trimetazidine normalizes postischemic function and fractional glucose oxidation in hypertrophied hearts, mainly by reducing glycolysis. These data extend the potential usefulness of trimetazidine and provide support for its use as a means to improve postischemic function of pressure overload hypertrophied hearts.
Assuntos
Cardiomegalia/fisiopatologia , Isquemia Miocárdica/fisiopatologia , Trimetazidina/farmacologia , Vasodilatadores/farmacologia , Trifosfato de Adenosina/biossíntese , Animais , Circulação Coronária/efeitos dos fármacos , Glucose/metabolismo , Glicólise/efeitos dos fármacos , Cinética , Ácido Láctico/metabolismo , Masculino , Contração Miocárdica/efeitos dos fármacos , Miocárdio/metabolismo , Oxirredução , Palmitatos/metabolismo , Perfusão , Prótons , Ratos , Ratos Sprague-DawleyRESUMO
Women less than 50 years of age, the majority of whom are likely premenopausal and exposed to estrogen, are at greater risk of a poor short-term recovery after myocardial ischemia than men and older women. Since estrogen enhances non-cardiac lipid utilization and increased lipid utilization is associated with poor post-ischemic heart function, we determined the effect of estrogen replacement on post-ischemic myocardial function and fatty acid oxidation. Female Sprague-Dawley rats, either intact (n = 15) or ovariectomized and treated with 17beta-estradiol (0.1 mg x kg(-1) x day(-1), s.c., n = 14) or corn oil vehicle (n = 16) for 5 weeks, were compared. Function and fatty acid oxidation of isolated working hearts perfused with 1.2 mM [9,10-3H]palmitate, 5.5 mM glucose, 0.5 mM lactate, and 100 mU/L insulin were measured before and after global no-flow ischemia. Only 36% of hearts from estrogen-treated rats recovered after ischemia compared with 56% from vehicle-treated rats (p > 0.05, not significant), while 93% of hearts from intact rats recovered (p < 0.05). Relative to pre-ischemic values, post-ischemic function of estrogen-treated hearts (26.3 +/- 10.1%) was significantly lower than vehicle-treated hearts (53.4 +/- 11.8%, p < 0.05) and hearts from intact rats (81.9 +/- 7.0%, p < 0.05). Following ischemia, fatty acid oxidation was greater in estrogen-treated hearts than in the other groups. Thus, estrogen replacement stimulates fatty acid oxidation and impairs post-ischemic recovery of isolated working hearts from ovariectomized female rats.
Assuntos
Estradiol/farmacologia , Ácidos Graxos/metabolismo , Isquemia Miocárdica/fisiopatologia , Miocárdio/metabolismo , Animais , Peso Corporal/efeitos dos fármacos , Estrogênios/sangue , Feminino , Coração/efeitos dos fármacos , Coração/fisiopatologia , Técnicas In Vitro , Isquemia Miocárdica/metabolismo , Reperfusão Miocárdica , Tamanho do Órgão/efeitos dos fármacos , Ovariectomia , Oxirredução , Ratos , Ratos Sprague-DawleyRESUMO
We tested the hypothesis that activation of AMP-activated protein kinase (AMPK) promotes myocardial glycogenolysis by decreasing glycogen synthase (GS) and/or increasing glycogen phosphorylase (GP) activities. Isolated working hearts from halothane-anesthetized male Sprague-Dawley rats perfused in the absence or presence of 0.8 or 1.2 mM 5-aminoimidazole-4-carboxamide 1-beta-d-ribofuranoside (AICAR), an adenosine analog and cell-permeable activator of AMPK, were studied. Glycogen degradation was increased by AICAR, while glycogen synthesis was not affected. AICAR increased myocardial 5-aminoimidazole-4-carboxamide 1-beta-d-ribofuranotide (ZMP), the active intracellular form of AICAR, but did not alter the activity of GS and GP measured in tissue homogenates or the content of glucose-6-phosphate and adenine nucleotides in freeze-clamped tissue. Importantly, the calculated intracellular concentration of ZMP achieved in this study was similar to the K(m) value of ZMP for GP determined in homogenates of myocardial tissue. We conclude that the data are consistent with allosteric activation of GP by ZMP being responsible for the glycogenolysis caused by AICAR in the intact rat heart.
Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Glicogênio/metabolismo , Coração/efeitos dos fármacos , Miocárdio/metabolismo , Ribonucleotídeos/farmacologia , Adenilato Quinase/metabolismo , Regulação Alostérica , Aminoimidazol Carboxamida/metabolismo , Animais , Ativação Enzimática/efeitos dos fármacos , Glicogênio Fosforilase/metabolismo , Glicogênio Sintase/antagonistas & inibidores , Glicogênio Sintase/metabolismo , Masculino , Miocárdio/enzimologia , Ratos , Ratos Sprague-Dawley , Ribonucleotídeos/metabolismo , Estereoisomerismo , Fatores de TempoRESUMO
Glycolysis, measured by (3)H(2)O production from [5-(3)H]glucose, is accelerated in isolated working hypertrophied rat hearts. However, nonglycolytic detritiation of [5-(3)H]glucose via the nonoxidative pentose phosphate pathway (PPP) could potentially lead to an overestimation of true glycolytic rates, especially in hypertrophied hearts where the PPP may be upregulated. To address this concern, we measured glycolysis using [5-(3)H]glucose and a second, independent method in isolated working hearts from halothane-anesthetized, sham-operated and aortic-constricted rats. Glycolysis was accelerated in hypertrophied hearts compared with control hearts regardless of the method used. There was also excellent concordance in glycolytic rates between the different methods. Moreover, activity of glucose-6-phosphate dehydrogenase and expression of transaldolase, enzymes controlling key steps in the oxidative and nonoxidative PPP, respectively, were not different between control and hypertrophied hearts. Thus nonglycolytic detritiation of [5-(3)H]glucose in the PPP is insignificant, and (3)H(2)O production from [5-(3)H]glucose is an accurate means to measure glycolysis in isolated working normal and hypertrophied rat hearts. Furthermore, the PPP does not appear to be increased in cardiac hypertrophy induced by abdominal aortic constriction.
Assuntos
Artefatos , Glicólise/fisiologia , Hipertrofia Ventricular Esquerda/metabolismo , Animais , Pressão Sanguínea , Glucose/farmacocinética , Glucosefosfato Desidrogenase/metabolismo , Frequência Cardíaca , Hipertrofia Ventricular Esquerda/patologia , Hipertrofia Ventricular Esquerda/fisiopatologia , Ácido Láctico/metabolismo , Masculino , Miocárdio/enzimologia , Miocárdio/patologia , Tamanho do Órgão , Oxirredução , Via de Pentose Fosfato/fisiologia , Ácido Pirúvico/metabolismo , Ratos , Ratos Sprague-Dawley , Transaldolase/metabolismo , TrítioRESUMO
Diabetic patients are particularly susceptible to cardiomyopathy independent of vascular disease, and recent evidence implicates cell death as a contributing factor. Given its protective role against apoptosis, we hypothesized that dietary n-6 polyunsaturated fatty acid (PUFA) may well decrease the incidence of this mode of cardiac cell death after diabetes. Male Wistar rats were first fed a diet rich in n-6 PUFA [20% (wt/wt) sunflower oil] for 4 wk followed by streptozotocin (STZ, 55 mg/kg) to induce diabetes. After a brief period of hyperglycemia (4 days), hearts were excised for functional, morphological, and biochemical analysis. In diabetic rats, n-6 PUFA decreased caspase-3 activity, crucial for myocardial apoptosis. However, cardiac necrosis, an alternative mode of cell death, increased. In these hearts, a rise in linoleic acid and depleted cardiac glutathione could explain this "switch" to necrotic cell death. Additionally, mitochondrial abnormalities, impaired substrate utilization, and enhanced triglyceride accumulation could have also contributed to a decline in cardiac function in these animals. Our study provides evidence that, in contrast to other models of diabetic cardiomyopathy that exhibit cardiac dysfunction only after chronic hyperglycemia, n-6 PUFA feeding coupled with only 4 days of diabetes precipitated metabolic and contractile abnormalities in the heart. Thus, although promoted as being beneficial, excess n-6 PUFA, with its predisposition to induce obesity, insulin resistance, and ultimately diabetes, could accelerate myocardial abnormalities in diabetic patients.
Assuntos
Diabetes Mellitus Experimental/patologia , Ácidos Graxos Ômega-6/farmacocinética , Hiperglicemia/patologia , Miocárdio/metabolismo , Miocárdio/patologia , Ração Animal , Animais , Caspase 3 , Caspases/metabolismo , Morte Celular/efeitos dos fármacos , Diabetes Mellitus Experimental/metabolismo , Gorduras na Dieta/farmacologia , Glucose/metabolismo , Testes de Função Cardíaca , Hiperglicemia/metabolismo , Masculino , Microscopia Eletrônica , Mitocôndrias/patologia , Mitocôndrias/ultraestrutura , Necrose , Oxirredução , Palmitatos/metabolismo , Óleos de Plantas/farmacocinética , Ratos , Ratos Wistar , Óleo de GirassolRESUMO
Adaptation of myocardial energy substrate utilization may contribute to the cardioprotective effects of regular exercise, a possibility supported by evidence showing that pharmacological metabolic modulation is beneficial to ischemic hearts during reperfusion. Thus we tested the hypothesis that the beneficial effect of regular physical exercise on recovery from ischemia-reperfusion is associated with a protective metabolic phenotype. Function, glycolysis, and oxidation of glucose, lactate, and palmitate were measured in isolated working hearts from sedentary control (C) and treadmill-trained (T: 10 wk, 4 days/wk) female Sprague-Dawley rats submitted to 20 min ischemia and 40 min reperfusion. Training resulted in myocardial hypertrophy (1.65 +/- 0.05 vs. 1.30 +/- 0.03 g heart wet wt, P < 0.001) and improved recovery of function after ischemia by nearly 50% (P < 0.05). Glycolysis was 25-30% lower in T hearts before and after ischemia (P < 0.05), whereas rates of glucose oxidation were 45% higher before ischemia (P < 0.01). As a result, the fraction of glucose oxidized before and after ischemia was, respectively, twofold and 25% greater in T hearts (P < 0.05). Palmitate oxidation was 50-65% greater in T than in C before and after ischemia (P < 0.05), whereas lactate oxidation did not differ between groups. Alteration in content of selected enzymes and proteins, as assessed by immunoblot analysis, could not account for the reduction in glycolysis or increase in glucose and palmitate oxidation observed. Combined with the studies on the beneficial effect of pharmacological modulation of energy metabolism, the present results provide support for a role of metabolic adaptations in protecting the trained heart against ischemia-reperfusion injury.