RESUMO
(1) Background: The exact mechanism(s) underlying pathological changes in a heart in transition to hypertrophy and failure are not yet fully understood. However, alterations in cardiac energy metabolism seem to be an important contributor. We characterized an in vitro model of adrenergic stimulation-induced cardiac hypertrophy for studying metabolic, structural, and functional changes over time. Accordingly, we investigated whether metabolic interventions prevent cardiac structural and functional changes; (2) Methods: Primary rat cardiomyocytes were treated with phenylephrine (PE) for 16 h, 24 h, or 48 h, whereafter hypertrophic marker expression, protein synthesis rate, glucose uptake, and contractile function were assessed; (3) Results: 24 h PE treatment increased expression of hypertrophic markers, phosphorylation of hypertrophy-related signaling kinases, protein synthesis, and glucose uptake. Importantly, the increased glucose uptake preceded structural and functional changes, suggesting a causal role for metabolism in the onset of PE-induced hypertrophy. Indeed, PE treatment in the presence of a PAN-Akt inhibitor or of a GLUT4 inhibitor dipyridamole prevented PE-induced increases in cellular glucose uptake and ameliorated PE-induced contractile alterations; (4) Conclusions: Pharmacological interventions, forcing substrate metabolism away from glucose utilization, improved contractile properties in PE-treated cardiomyocytes, suggesting that targeting glucose uptake, independent from protein synthesis, forms a promising strategy to prevent hypertrophy and hypertrophy-induced cardiac dysfunction.
Assuntos
Cardiomegalia/metabolismo , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/fisiologia , Animais , Animais Recém-Nascidos , Células Cultivadas , Metabolismo Energético , Glucose/metabolismo , Contração Muscular/efeitos dos fármacos , Contração Muscular/fisiologia , Miócitos Cardíacos/efeitos dos fármacos , Fenilefrina/farmacologia , Fosforilação , Ratos , Transdução de Sinais/efeitos dos fármacosRESUMO
Small heterodimer partner (SHP) is an atypical nuclear receptor expressed in heart that has been shown to inhibit the hypertrophic response. Here, we assessed the role of SHP in cardiac metabolism and inflammation. Mice fed a high-fat diet (HFD) displayed glucose intolerance accompanied by increased cardiac mRNA levels of Shp. In HL-1 cardiomyocytes, SHP overexpression inhibited both basal and insulin-stimulated glucose uptake and impaired the insulin signalling pathway (evidenced by reduced AKT and AS160 phosphorylation), similar to insulin resistant cells generated by high palmitate/high insulin treatment (HP/HI; 500µM/100nM). In addition, SHP overexpression increased Socs3 mRNA and reduced IRS-1 protein levels. SHP overexpression also induced Cd36 expression (~6.2 fold; p<0.001) linking to the observed intramyocellular lipid accumulation. SHP overexpressing cells further showed altered expression of genes involved in lipid metabolism, i.e., Acaca, Acadvl or Ucp3, augmented NF-κB DNA-binding activity and induced transcripts of inflammatory genes, i.e., Il6 and Tnf mRNA (~4-fold induction, p<0.01). Alterations in metabolism and inflammation found in SHP overexpressing cells were associated with changes in the mRNA levels of Ppara (79% reduction, p<0.001) and Pparg (~58-fold induction, p<0.001). Finally, co-immunoprecipitation studies showed that SHP overexpression strongly reduced the physical interaction between PPARα and the p65 subunit of NF-κB, suggesting that dissociation of these two proteins is one of the mechanisms by which SHP initiates the inflammatory response in cardiac cells. Overall, our results suggest that SHP upregulation upon high-fat feeding leads to lipid accumulation, insulin resistance and inflammation in cardiomyocytes.
Assuntos
Inflamação/metabolismo , Resistência à Insulina/genética , Metabolismo dos Lipídeos/genética , Miocárdio/metabolismo , Receptores Citoplasmáticos e Nucleares/biossíntese , Animais , Regulação da Expressão Gênica , Glucose/metabolismo , Humanos , Inflamação/patologia , Insulina/metabolismo , Camundongos , Miocárdio/patologia , Miócitos Cardíacos/metabolismo , NF-kappa B/genética , NF-kappa B/metabolismo , RNA Mensageiro/biossíntese , RNA Mensageiro/metabolismo , Receptores Citoplasmáticos e Nucleares/genética , Transdução de SinaisRESUMO
AMP-activated protein kinase (AMPK) is a metabolic stress-sensing kinase. We previously showed that glucose deprivation induces autophosphorylation of AMPKß at Thr-148, which prevents the binding of AMPK to glycogen. Furthermore, in MIN6 cells, AMPKß1 binds to R6 (PPP1R3D), a glycogen-targeting subunit of protein phosphatase type 1 (PP1), thereby regulating the glucose-induced inactivation of AMPK. In the present study, we further investigated the interaction of R6 with AMPKß and the possible dependency on Thr-148 phosphorylation status. Yeast two-hybrid (Y2H) analyses and co-immunoprecipitation (IP) of the overexpressed proteins in human embryonic kidney (HEK) 293T) cells revealed that both AMPKß1 and AMPK-ß2 wild-type (WT) isoforms bind to R6. The AMPKß-R6 interaction was stronger with the muscle-specific AMPKß2-WT and required association with the substrate-binding motif of R6. When HEK293T cells or C2C12 myotubes were cultured in high-glucose medium, AMPKß2-WT and R6 weakly interacted. In contrast, glycogen depletion significantly enhanced this protein interaction. Mutation of AMPKß2 Thr-148 prevented the interaction with R6 irrespective of the intracellular glycogen content. Treatment with the AMPK activator oligomycin enhanced the AMPKß2-R6 interaction in conjunction with increased Thr-148 phosphorylation in cells grown in low-glucose medium. These data are in accordance with R6 binding directly to AMPKß2 when both proteins detach from the diminishing glycogen particle, which is simultaneous with increased AMPKß2 Thr-148 autophosphorylation. Such a model points to a possible control of AMPK by PP1-R6 upon glycogen depletion in muscle.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Glicogênio/metabolismo , Mutação de Sentido Incorreto , Proteína Fosfatase 1/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Substituição de Aminoácidos , Glicogênio/genética , Células HEK293 , Humanos , Fosforilação , Ligação Proteica , Proteína Fosfatase 1/genéticaRESUMO
Insulin resistance is an important risk factor for the development of several cardiac pathologies, thus advocating strategies for restoring insulin sensitivity of the heart in these conditions. Omega-3 polyunsaturated fatty acids (ω-3 PUFAs), mainly eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3), have been shown to improve insulin sensitivity in insulin-sensitive tissues, but their direct effect on insulin signaling and metabolic parameters in the myocardium has not been reported previously. The aim of this study was therefore to examine the ability of EPA and DHA to prevent insulin resistance in isolated rat cardiomyocytes. Primary rat cardiomyocytes were made insulin resistant by 48 h incubation in high insulin (HI) medium. Parallel incubations were supplemented by 200 µM EPA or DHA. Addition of EPA or DHA to the medium prevented the induction of insulin resistance in cardiomyocytes by preserving the phosphorylation state of key proteins in the insulin signaling cascade and by preventing persistent relocation of fatty acid transporter CD36 to the sarcolemma. Only cardiomyocytes incubated in the presence of EPA, however, exhibited improvements in glucose and fatty acid uptake and cell shortening. We conclude that ω-3 PUFAs protect metabolic and functional properties of cardiomyocytes subjected to insulin resistance-evoking conditions.
Assuntos
Cardiotônicos/farmacologia , Ácidos Docosa-Hexaenoicos/farmacologia , Ácido Eicosapentaenoico/farmacologia , Metabolismo Energético/efeitos dos fármacos , Resistência à Insulina , Insulina/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Animais , Antígenos CD36/metabolismo , Células Cultivadas , Ácidos Docosa-Hexaenoicos/metabolismo , Ácido Eicosapentaenoico/metabolismo , Glucose/metabolismo , Masculino , Contração Miocárdica/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Fosforilação , Transporte Proteico , Ratos Endogâmicos Lew , Sarcolema/efeitos dos fármacos , Sarcolema/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de TempoRESUMO
In the insulin resistant heart, energy fuel selection shifts away from glucose utilization towards almost complete dependence on long-chain fatty acids (LCFA). This shift results in excessive cardiac lipid accumulation and eventually heart failure. Lipid-induced cardiomyopathy may be averted by strategies that increase glucose uptake without elevating LCFA uptake. Protein kinase-D1 (PKD1) is involved in contraction-induced glucose, but not LCFA, uptake allowing to hypothesize that this kinase is an attractive target to treat lipid-induced cardiomyopathy. For this, cardiospecific constitutively active PKD1 overexpression (caPKD1)-mice were subjected to an insulin resistance-inducing high fat-diet for 20-weeks. Substrate utilization was assessed by microPET and cardiac function by echocardiography. Cardiomyocytes were isolated for measurement of substrate uptake, lipid accumulation and insulin sensitivity. Wild-type mice on a high fat-diet displayed increased basal myocellular LCFA uptake, increased lipid deposition, greatly impaired insulin signaling, and loss of insulin-stimulated glucose and LCFA uptake, which was associated with concentric hypertrophic remodeling. The caPKD1 mice on high-fat diet showed none of these characteristics, whereas on low-fat diet a shift towards cardiac glucose utilization in combination with hypertrophy and ventricular dilation was observed. In conclusion, these data suggest that PKD pathway activation may be an attractive therapeutic strategy to mitigate lipid accumulation, insulin resistance and maladaptive remodeling in the lipid-overloaded heart, but this requires further investigation.
Assuntos
Cardiomiopatia Dilatada/enzimologia , Resistência à Insulina , Proteína Quinase C/metabolismo , Animais , Dieta Hiperlipídica/efeitos adversos , Feminino , Expressão Gênica , Ventrículos do Coração/metabolismo , Ventrículos do Coração/patologia , Histona Desacetilases/metabolismo , Metabolismo dos Lipídeos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Miocárdio/enzimologia , Miocárdio/patologia , Miócitos Cardíacos/enzimologia , Fosforilação , Proteína Quinase C/genética , Processamento de Proteína Pós-TraducionalRESUMO
Activation of AMP-activated protein kinase (AMPK) in cardiomyocytes induces translocation of glucose transporter GLUT4 and long-chain fatty acid (LCFA) transporter CD36 from endosomal stores to the sarcolemma to enhance glucose and LCFA uptake, respectively. Ca(2+)/calmodulin-activated kinase kinase-ß (CaMKKß) has been positioned directly upstream of AMPK. However, it is unknown whether acute increases in [Ca(2+)]i stimulate translocation of GLUT4 and CD36 and uptake of glucose and LCFA or whether Ca(2+) signaling converges with AMPK signaling to exert these actions. Therefore, we studied the interplay between Ca(2+) and AMPK signaling in regulation of cardiomyocyte substrate uptake. Exposure of primary cardiomyocytes to inhibitors or activators of Ca(2+) signaling affected neither AMPK-Thr(172) phosphorylation nor basal and AMPK-mediated glucose and LCFA uptake. Despite their lack of an effect on substrate uptake, Ca(2+) signaling activators induced GLUT4 and CD36 translocation. In contrast, AMPK activators stimulated GLUT4/CD36 translocation as well as glucose/LCFA uptake. When cardiomyocytes were cotreated with Ca(2+) signaling and AMPK activators, Ca(2+) signaling activators further enhanced AMPK-induced glucose/LCFA uptake. In conclusion, Ca(2+) signaling shows no involvement in AMPK-induced GLUT4/CD36 translocation and substrate uptake but elicits transporter translocation via a separate pathway requiring CaMKKß/CaMKs. Ca(2+)-induced transporter translocation by itself appears to be ineffective to increase substrate uptake but requires additional AMPK activation to effectuate transporter translocation into increased substrate uptake. Ca(2+)-induced transporter translocation might be crucial under excessive cardiac stress conditions that require supraphysiological energy demands. Alternatively, Ca(2+) signaling might prepare the heart for substrate uptake during physiological contraction by inducing transporter translocation.
Assuntos
Antígenos CD36/metabolismo , Sinalização do Cálcio/fisiologia , Ácidos Graxos/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Glucose/metabolismo , Miócitos Cardíacos/metabolismo , Sarcolema/metabolismo , Animais , Calcimicina/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Miócitos Cardíacos/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , Ratos , Ratos Endogâmicos Lew , Sarcolema/efeitos dos fármacos , Tapsigargina/farmacologiaRESUMO
Increased contraction enhances substrate uptake into cardiomyocytes via translocation of the glucose transporter GLUT4 and the long chain fatty acid (LCFA) transporter CD36 from intracellular stores to the sarcolemma. Additionally, contraction activates the signaling enzymes AMP-activated protein kinase (AMPK) and protein kinase D1 (PKD1). Although AMPK has been implicated in contraction-induced GLUT4 and CD36 translocation in cardiomyocytes, the precise role of PKD1 in these processes is not known. To study this, we triggered contractions in cardiomyocytes by electric field stimulation (EFS). First, the role of PKD1 in GLUT4 and CD36 translocation was defined. In PKD1 siRNA-treated cardiomyocytes as well as cardiomyocytes from PKD1 knock-out mice, EFS-induced translocation of GLUT4, but not CD36, was abolished. In AMPK siRNA-treated cardiomyocytes and cardiomyocytes from AMPKα2 knock-out mice, both GLUT4 and CD36 translocation were abrogated. Hence, unlike AMPK, PKD1 is selectively involved in glucose uptake. Second, we analyzed upstream factors in PKD1 activation. Cardiomyocyte contractions enhanced reactive oxygen species (ROS) production. Using ROS scavengers, we found that PKD1 signaling and glucose uptake are more sensitive to changes in intracellular ROS than AMPK signaling or LCFA uptake. Furthermore, silencing of death-activated protein kinase (DAPK) abrogated EFS-induced GLUT4 but not CD36 translocation. Finally, possible links between PKD1 and AMPK signaling were investigated. PKD1 silencing did not affect AMPK activation. Reciprocally, AMPK silencing did not alter PKD1 activation. In conclusion, we present a novel contraction-induced ROS-DAPK-PKD1 pathway in cardiomyocytes. This pathway is activated separately from AMPK and mediates GLUT4 translocation/glucose uptake, but not CD36 translocation/LCFA uptake.
Assuntos
Ácidos Graxos/metabolismo , Glucose/metabolismo , Contração Muscular , Miócitos Cardíacos/metabolismo , Proteína Quinase C/metabolismo , Proteínas Quinases Ativadas por AMP/deficiência , Proteínas Quinases Ativadas por AMP/genética , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Antígenos CD36/metabolismo , Linhagem Celular , Feminino , Técnicas de Silenciamento de Genes , Transportador de Glucose Tipo 4/metabolismo , Masculino , Camundongos , Miócitos Cardíacos/citologia , Proteína Quinase C/deficiência , Proteína Quinase C/genética , Transporte Proteico , Ratos , Espécies Reativas de Oxigênio/metabolismo , Transdução de SinaisRESUMO
Cardiac glucose utilization is regulated by reversible translocation of the glucose transporter GLUT4 from intracellular stores to the plasma membrane. During the onset of diet-induced insulin resistance, elevated lipid levels in the circulation interfere with insulin-stimulated GLUT4 translocation, leading to impaired glucose utilization. Recently, we identified vesicle-associated membrane protein (VAMP) 2 and 3 to be required for insulin- and contraction-stimulated GLUT4 translocation, respectively, in cardiomyocytes. Here, we investigated whether overexpression of VAMP2 and/or VAMP3 could protect insulin-stimulated GLUT4 translocation under conditions of insulin resistance. HL-1 atrial cardiomyocytes transiently overexpressing either VAMP2 or VAMP3 were cultured for 16 h with elevated concentrations of palmitate and insulin. Upon subsequent acute stimulation with insulin, we measured GLUT4 translocation, plasmalemmal presence of the fatty acid transporter CD36, and myocellular lipid accumulation. Overexpression of VAMP3, but not VAMP2, completely prevented lipid-induced inhibition of insulin-stimulated GLUT4 translocation. Furthermore, the plasmalemmal presence of CD36 and intracellular lipid levels remained normal in cells overexpressing VAMP3. However, insulin signaling was not retained, indicating an effect of VAMP3 overexpression downstream of PKB/Akt. Furthermore, we revealed that endogenous VAMP3 is bound by the contraction-activated protein kinase D (PKD), and contraction and VAMP3 overexpression protect insulin-stimulated GLUT4 translocation via a common mechanism. These observations indicate that PKD activates GLUT4 translocation via a VAMP3-dependent trafficking step, which pathway might be valuable to rescue constrained glucose utilization in the insulin-resistant heart.
Assuntos
Transportador de Glucose Tipo 4/metabolismo , Resistência à Insulina , Miócitos Cardíacos/metabolismo , Proteína 2 Associada à Membrana da Vesícula/metabolismo , Proteína 3 Associada à Membrana da Vesícula/metabolismo , Animais , Antígenos CD36/metabolismo , Linhagem Celular , Gorduras na Dieta/farmacologia , Expressão Gênica , Cardiopatias/metabolismo , Cardiopatias/patologia , Insulina/farmacologia , Insulina/fisiologia , Metabolismo dos Lipídeos , Masculino , Camundongos , Contração Miocárdica , Miócitos Cardíacos/patologia , Miócitos Cardíacos/fisiologia , Palmitatos/farmacologia , Proteína Quinase C/metabolismo , Transporte Proteico , Ratos , Ratos Endogâmicos Lew , Transdução de Sinais , Proteína 2 Associada à Membrana da Vesícula/genética , Proteína 3 Associada à Membrana da Vesícula/genéticaRESUMO
An increased cardiac fatty acid supply and increased sarcolemmal presence of the long-chain fatty acid transporter CD36 are associated with and contribute to impaired cardiac insulin sensitivity and function. In the present study we aimed at preventing the development of insulin resistance and contractile dysfunction in cardiomyocytes by blocking CD36-mediated palmitate uptake. Insulin resistance and contractile dysfunction were induced in primary cardiomyocytes by 48 h incubation in media containing either 100 nM insulin (high insulin; HI) or 200 µM palmitate (high palmitate; HP). Under both culture conditions, insulin-stimulated glucose uptake and Akt phosphorylation were abrogated or markedly reduced. Furthermore, cardiomyocytes cultured in each medium displayed elevated sarcolemmal CD36 content, increased basal palmitate uptake, lipid accumulation and decreased sarcomere shortening. Immunochemical CD36 inhibition enhanced basal glucose uptake and prevented elevated basal palmitate uptake, triacylglycerol accumulation and contractile dysfunction in cardiomyocytes cultured in either medium. Additionally, CD36 inhibition prevented loss of insulin signalling in cells cultured in HP, but not in HI medium. In conclusion, CD36 inhibition prevents lipid accumulation and lipid-induced contractile dysfunction in cardiomyocytes, but probably independently of effects on insulin signalling. Nonetheless, pharmacological CD36 inhibition may be considered as a treatment strategy to counteract impaired functioning of the lipid-loaded heart.
Assuntos
Antígenos CD36/fisiologia , Resistência à Insulina/fisiologia , Miócitos Cardíacos/metabolismo , Palmitatos/metabolismo , Animais , Transporte Biológico , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Células Cultivadas/metabolismo , Cardiomiopatias Diabéticas/etiologia , Cardiomiopatias Diabéticas/metabolismo , Cardiomiopatias Diabéticas/prevenção & controle , Ácidos Graxos/metabolismo , Glucose/metabolismo , Insulina/farmacologia , Masculino , Mitocôndrias Cardíacas/metabolismo , Contração Miocárdica , Miócitos Cardíacos/efeitos dos fármacos , Palmitatos/farmacologia , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Endogâmicos Lew , Sarcolema/metabolismo , Sarcômeros/ultraestrutura , Transdução de Sinais/efeitos dos fármacos , Triglicerídeos/metabolismoRESUMO
Cardiac patients often are obese and have hypertension, but in most studies these conditions are investigated separately. Here, we aimed at 1) elucidating the interaction of metabolic and mechanophysical stress in the development of cardiac dysfunction in mice and 2) preventing this interaction by ablation of the fatty acid transporter CD36. Male wild-type (WT) C57Bl/6 mice and CD36(-/-) mice received chow or Western-type diet (WTD) for 10 wk and then underwent a sham surgery or transverse aortic constriction (TAC) under anesthesia. After a 6-wk continuation of the diet, cardiac function, morphology, lipid profiles, and molecular parameters were assessed. WTD administration affected body and organ weights of WT and CD36(-/-) mice, but it affected only plasma glucose and insulin concentrations in WT mice. Cardiac lipid concentrations increased in WT mice receiving WTD, decreased in CD36(-/-) on chow, and remained unchanged in CD36(-/-) receiving WTD. TAC induced cardiac hypertrophy in WT mice on chow but did not affect cardiac function and cardiac lipid concentrations. WTD or CD36 ablation worsened the outcome of TAC. Ablation of CD36 protected against the WTD-related aggravation of cardiac functional and structural changes induced by TAC. In conclusion, cardiac dysfunction and remodeling worsen when the heart is exposed to two stresses, metabolic and mechanophysical, at the same time. CD36 ablation prevents the metabolic stress resulting from a WTD. Thus, metabolic conditions are a critical factor for the compromised heart and provide new targets for metabolic manipulation in cardioprotection.
Assuntos
Antígenos CD36/genética , Cardiomegalia/metabolismo , Miocárdio/metabolismo , Animais , Estenose da Valva Aórtica/complicações , Glicemia/metabolismo , Antígenos CD36/metabolismo , Cardiomegalia/etiologia , Cardiomegalia/fisiopatologia , Dieta , Coração/fisiopatologia , Masculino , Camundongos , Camundongos Knockout , Miocárdio/patologia , Obesidade/complicaçõesRESUMO
Insulin and contraction stimulate both cardiac glucose and long-chain fatty acid (LCFA) uptake via translocation of the substrate transporters GLUT4 and CD36, respectively, from intracellular compartments to the sarcolemma. Little is known about the role of vesicular trafficking elements in insulin- and contraction-stimulated glucose and LCFA uptake in the heart, especially whether certain trafficking elements are specifically involved in GLUT4 versus CD36 translocation. Therefore, we studied the role of coat proteins, actin- and microtubule-filaments and endosomal pH on glucose and LCFA uptake into primary cardiomyocytes under basal conditions and during stimulation with insulin or oligomycin (contraction-like AMP-activated protein kinase activator). Inhibition of coat protein targeting to Golgi/endosomes decreased insulin/oligomycin-stimulated glucose (-42%/-51%) and LCFA (-39%/-68%) uptake. Actin disruption decreased insulin/oligomycin-stimulated glucose uptake (-41%/-75%), while not affecting LCFA uptake. Microtubule disruption did not affect substrate uptake under any condition. Endosomal alkalinization increased basal sarcolemmal CD36 (2-fold), but not GLUT4, content, and concomitantly decreased basal intracellular membrane GLUT4 and CD36 content (-60% and -62%, respectively), indicating successful CD36 translocation and incomplete GLUT4 translocation. Additionally, endosomal alkalinization elevated basal LCFA uptake (1.4-fold) in a nonadditive manner to insulin/oligomycin, and decreased insulin/oligomycin-stimulated glucose uptake (-32%/-68%). In conclusion, 1) CD36 translocation, just like GLUT4 translocation, is a vesicle-mediated process depending on coat proteins, and 2) GLUT4 and CD36 trafficking are differentially dependent on endosomal pH and actin filaments. The latter conclusion suggests novel strategies to alter cardiac substrate preference as part of metabolic modulation therapy.
Assuntos
Citoesqueleto de Actina/metabolismo , Antígenos CD36/metabolismo , Desoxiglucose/metabolismo , Endossomos/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Miócitos Cardíacos/metabolismo , Ácido Palmítico/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/ultraestrutura , Animais , Transporte Biológico , Brefeldina A/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Proteína Coatomer/metabolismo , Colchicina/farmacologia , Endossomos/efeitos dos fármacos , Endossomos/ultraestrutura , Ativação Enzimática , Ativadores de Enzimas/farmacologia , Concentração de Íons de Hidrogênio , Insulina/metabolismo , Masculino , Contração Miocárdica , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/ultraestrutura , Oligomicinas/farmacologia , Transporte Proteico , Ratos , Ratos Endogâmicos Lew , Tiazolidinas/farmacologia , Moduladores de Tubulina/farmacologiaRESUMO
Enhanced contractile activity increases cardiac long-chain fatty acid (LCFA) uptake via translocation of CD36 to the sarcolemma, similarly to increase in glucose uptake via GLUT4 translocation. AMP-activated protein kinase (AMPK) is assumed to mediate contraction-induced LCFA utilization. However, which catalytic isoform (AMPKalpha1 versus AMPKalpha2) is involved, is unknown. Furthermore, no studies have been performed on the role of LKB1, a kinase with AMPKK activity, on the regulation of cardiac LCFA utilization. Using different mouse models (AMPKalpha2-kinase-dead, AMPKalpha2-knockout and LKB1-knockout mice), we tested whether LKB1 and/or AMPK are required for stimulation of LCFA and glucose utilization upon treatment of cardiomyocytes with compounds (oligomycin/AICAR/dipyridamole) which induce CD36 translocation similar to that seen upon contraction. In AMPKalpha2- kinase-dead cardiomyocytes, the stimulating effects of oligomycin and AICAR on palmitate and deoxyglucose uptake and palmitate oxidation were almost completely lost. Moreover, in AMPKalpha2- and LKB1-knockout cardiomyocytes, oligomycin-induced LCFA and deoxyglucose uptake were completely abolished. However, the stimulatory effect of dipyridamole on palmitate uptake and oxidation was preserved in AMPKalpha2-kinase-dead cardiomyocytes. In conclusion, in the heart there is a signaling axis consisting of LKB1 and AMPKalpha2 which activation results in enhanced LCFA utilization, similarly to enhanced glucose uptake. In addition, an unknown dipyridamole-activated pathway can stimulate cardiac LCFA utilization by activating signaling components downstream of AMPK.
Assuntos
Proteínas Quinases Ativadas por AMP/fisiologia , Antígenos CD36/metabolismo , Ácidos Graxos não Esterificados/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/farmacologia , Animais , Transporte Biológico , Desoxiglucose/metabolismo , Dipiridamol/farmacologia , Transportador de Glucose Tipo 4/metabolismo , Hipoglicemiantes/farmacologia , Integrases/metabolismo , Camundongos , Camundongos Knockout , Miócitos Cardíacos/citologia , Miócitos Cardíacos/efeitos dos fármacos , Oligomicinas/farmacologia , Oxirredução , Palmitatos/metabolismo , Fenótipo , Fosforilação , Inibidores da Agregação Plaquetária/farmacologia , Transporte Proteico , Ribonucleotídeos/farmacologia , Sarcolema/metabolismo , Desacopladores/farmacologiaRESUMO
Although CPT-I (carnitine palmitoyltransferase-I) is generally regarded to present a major rate-controlling site in mitochondrial beta-oxidation, it is incompletely understood whether CPT-I is rate-limiting in the overall LCFA (long-chain fatty acid) flux in the heart. Another important site of regulation of the LCFA flux in the heart is trans-sarcolemmal LCFA transport facilitated by CD36 and FABPpm (plasma membrane fatty acid-binding protein). Therefore, we explored to what extent a chronic pharmacological blockade of the LCFA flux at the level of mitochondrial entry of LCFA-CoA would affect sarcolemmal LCFA uptake. Rats were injected daily with saline or etomoxir, a specific CPT-I inhibitor, for 8 days at 20 mg/kg of body mass. Etomoxir-treated rats displayed a 44% reduced cardiac CPT-I activity. Sarcolemmal contents of CD36 and FABPpm, as well as the LCFA transport capacity, were not altered in the hearts of etomoxir-treated versus control rats. Furthermore, rates of LCFA uptake and oxidation, and glucose uptake by cardiac myocytes from etomoxir-treated rats were not different from control rats, neither under basal nor under acutely induced maximal metabolic demands. Finally, hearts from etomoxir-treated rats did not display triacylglycerol accumulation. Therefore CPT-I appears not to present a major rate-controlling site in total cardiac LCFA flux. It is likely that sarcolemmal LCFA entry rather than mitochondrial LCFA-CoA entry is a promising target for normalizing LCFA flux in cardiac metabolic diseases.
Assuntos
Carnitina O-Palmitoiltransferase/metabolismo , Compostos de Epóxi/farmacologia , Ácidos Graxos/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Western Blotting , Células Cultivadas , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática/efeitos dos fármacos , Masculino , Oxirredução/efeitos dos fármacos , Ratos , Triglicerídeos/metabolismoRESUMO
The role of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE)- and SNARE-associated proteins have not yet been assessed in regulation of cardiac glucose uptake, nor in the regulation of long-chain fatty acid (LCFA) uptake in any tissue. Munc18c is a SNARE-associated protein that regulates GLUT4 translocation in skeletal muscle and adipose tissue. Using cardiomyocytes from Munc18c(-/+) mice (with 56% reduction of Munc18c protein expression), we investigated whether this syntaxin4-associated protein is involved in regulation of cardiac substrate uptake. Basal, insulin- and oligomycin (a 5' AMP-activated protein kinase-activating agent)-stimulated glucose and LCFA uptake were not altered significantly in Munc18c(-/+) cardiomyocytes compared to wild-type cells. We conclude, therefore, that Munc18c is not rate-limiting for cardiac substrate uptake, neither under basal conditions nor when maximally stimulated metabolically.
Assuntos
Ácidos Graxos não Esterificados/metabolismo , Glucose/metabolismo , Proteínas Munc18/genética , Miócitos Cardíacos/metabolismo , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Transporte Biológico , Complexo CD3/metabolismo , Transportador de Glucose Tipo 4/genética , Transportador de Glucose Tipo 4/metabolismo , Insulina/análogos & derivados , Insulina/metabolismo , Insulina de Ação Prolongada , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas Munc18/metabolismo , Sarcolema/metabolismoRESUMO
Here we describe an assay for simultaneous measurement of cellular uptake rates of long-chain fatty acids (LCFA) and glucose that can be applied to cells in suspension. The uptake assay includes the use of radiolabeled substrates at such concentrations and incubation periods that exact information is provided about unidirectional uptakes rates. Cellular uptake of both substrates is under regulation of AMPK. The underlying mechanism includes the translocation of LCFA and glucose transporters from intracellular membrane compartments to the cell surface, leading to an increase in substrate uptake. In this chapter, we explain the principles of the uptake assay before detailing the exact procedure. We also provide information of the specific LCFA and glucose transporters subject to AMPK-mediated subcellular translocation. Finally, we discuss the application of AMPK inhibitors and activators in combination with cellular substrate uptake assays.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Ensaios Enzimáticos/métodos , Ácidos Graxos/metabolismo , Glucose/metabolismo , Animais , Células Cultivadas , Ensaios Enzimáticos/instrumentação , Transportador de Glucose Tipo 4/metabolismo , Membranas Intracelulares/metabolismo , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Cultura Primária de Células , RatosRESUMO
Dietary fat overconsumption leads to myocardial lipid accumulation through mechanisms that are incompletely resolved. Previously, we identified increased translocation of the fatty acid transporter CD36 from its endosomal storage compartment to the sarcolemma as the primary mechanism of excessive myocellular lipid import. Here, we show that increased CD36 translocation is caused by alkalinization of endosomes resulting from inhibition of proton pumping activity of vacuolar-type H+-ATPase (v-ATPase). Endosomal alkalinization was observed in hearts from rats fed a lard-based high-fat diet and in rodent and human cardiomyocytes upon palmitate overexposure, and appeared as an early lipid-induced event preceding the onset of insulin resistance. Either genetic or pharmacological inhibition of v-ATPase in cardiomyocytes exposed to low palmitate concentrations reduced insulin sensitivity and cardiomyocyte contractility, which was rescued by CD36 silencing. The mechanism of palmitate-induced v-ATPase inhibition involved its dissociation into two parts: the cytosolic V1 and the integral membrane V0 subcomplex. Interestingly, oleate also inhibits v-ATPase function, yielding triacylglycerol accumulation but not insulin resistance. In conclusion, lipid oversupply increases CD36-mediated lipid uptake that directly impairs v-ATPase function. This feeds forward to enhanced CD36 translocation and further increased lipid uptake. In the case of palmitate, its accelerated uptake ultimately precipitates into cardiac insulin resistance and contractile dysfunction.
Assuntos
Antígenos CD36/metabolismo , Endossomos/efeitos dos fármacos , Glucose/metabolismo , Coração/efeitos dos fármacos , Resistência à Insulina , Contração Miocárdica/efeitos dos fármacos , Miocárdio/metabolismo , Miócitos Cardíacos/efeitos dos fármacos , Palmitatos/farmacologia , ATPases Vacuolares Próton-Translocadoras/antagonistas & inibidores , Animais , Western Blotting , Radioisótopos de Carbono , Células Cultivadas , Desoxiglucose/metabolismo , Dieta Hiperlipídica , Endossomos/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Células-Tronco Pluripotentes Induzidas , Masculino , Miócitos Cardíacos/metabolismo , Cadeias Pesadas de Miosina/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Endogâmicos Lew , Triglicerídeos/metabolismo , Trítio , Troponina T/genéticaRESUMO
AIMS: The type 2 diabetic heart oxidizes more fat and less glucose, which can impair metabolic flexibility and function. Increased sarcolemmal fatty acid translocase (FAT/CD36) imports more fatty acid into the diabetic myocardium, feeding increased fatty acid oxidation and elevated lipid deposition. Unlike other metabolic modulators that target mitochondrial fatty acid oxidation, we proposed that pharmacologically inhibiting fatty acid uptake, as the primary step in the pathway, would provide an alternative mechanism to rebalance metabolism and prevent lipid accumulation following hypoxic stress. METHODS AND RESULTS: Hearts from type 2 diabetic and control male Wistar rats were perfused in normoxia, hypoxia and reoxygenation, with the FAT/CD36 inhibitor sulfo-N-succinimidyl oleate (SSO) infused 4 min before hypoxia. SSO infusion into diabetic hearts decreased the fatty acid oxidation rate by 29% and myocardial triglyceride concentration by 48% compared with untreated diabetic hearts, restoring fatty acid metabolism to control levels following hypoxia-reoxygenation. SSO infusion increased the glycolytic rate by 46% in diabetic hearts during hypoxia, increased pyruvate dehydrogenase activity by 53% and decreased lactate efflux rate by 56% compared with untreated diabetic hearts during reoxygenation. In addition, SSO treatment of diabetic hearts increased intermediates within the second span of the Krebs cycle, namely fumarate, oxaloacetate, and the FAD total pool. The cardiac dysfunction in diabetic hearts following decreased oxygen availability was prevented by SSO-infusion prior to the hypoxic stress. Infusing SSO into diabetic hearts increased rate pressure product by 60% during hypoxia and by 32% following reoxygenation, restoring function to control levels. CONCLUSIONS: Diabetic hearts have limited metabolic flexibility and cardiac dysfunction when stressed, which can be rapidly rectified by reducing fatty acid uptake with the FAT/CD36 inhibitor, SSO. This novel therapeutic approach not only reduces fat oxidation but also lipotoxicity, by targeting the primary step in the fatty acid metabolism pathway.
Assuntos
Antígenos CD36/antagonistas & inibidores , Diabetes Mellitus Tipo 2/complicações , Cardiomiopatias Diabéticas/tratamento farmacológico , Metabolismo Energético/efeitos dos fármacos , Metabolismo dos Lipídeos/efeitos dos fármacos , Traumatismo por Reperfusão Miocárdica/tratamento farmacológico , Miocárdio/metabolismo , Ácidos Oleicos/farmacologia , Sarcolema/efeitos dos fármacos , Succinimidas/farmacologia , Animais , Antígenos CD36/metabolismo , Hipóxia Celular , Ciclo do Ácido Cítrico/efeitos dos fármacos , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/fisiopatologia , Cardiomiopatias Diabéticas/etiologia , Cardiomiopatias Diabéticas/metabolismo , Cardiomiopatias Diabéticas/fisiopatologia , Ácidos Graxos/metabolismo , Preparação de Coração Isolado , Masculino , Traumatismo por Reperfusão Miocárdica/etiologia , Traumatismo por Reperfusão Miocárdica/metabolismo , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Oxirredução , Estresse Oxidativo/efeitos dos fármacos , Ratos Wistar , Sarcolema/metabolismo , Fatores de Tempo , Triglicerídeos/metabolismo , Função Ventricular Esquerda/efeitos dos fármacos , Pressão Ventricular/efeitos dos fármacosRESUMO
The protein thiol-modifying agent arsenite, a potent activator of stress signaling, was used to examine the involvement of MAPKs in the regulation of cardiac substrate uptake. Arsenite strongly induced p38 MAPK phosphorylation in isolated rat cardiac myocytes but also moderately enhanced phosphorylation of p42/44 ERK and p70 S6K. At the level of cardiomyocytic substrate use, arsenite enhanced glucose uptake dose dependently up to 5.1-fold but failed to stimulate long-chain fatty acid uptake. At the substrate transporter level, arsenite stimulated the translocation of GLUT4 to the sarcolemma but failed to recruit CD36 or FABPpm. Because arsenite did not influence the intrinsic activity of glucose transporters, GLUT4 translocation is entirely responsible for the selective increase in glucose uptake by arsenite. Moreover, the nonadditivity of arsenite-induced glucose uptake and insulin-induced glucose uptake indicates that arsenite recruits GLUT4 from insulin-responsive intracellular stores. Inhibitor studies with SB203580/SB202190, PD98059, and rapamycin indicate that activation of p38 MAPK, p42/44 ERK, and p70 S6K, respectively, are not involved in arsenite-induced glucose uptake. In addition, all these kinases do not play a role in regulation of cardiac glucose and long-chain fatty acid uptake by insulin. Hence, arsenite's selective stimulation of glucose uptake appears unrelated to its signaling actions, suggesting that arsenite acts via thiol modification of a putative intracellular protein target of arsenite within insulin-responsive GLUT4-containing stores. Because of arsenite's selective stimulation of cardiac glucose uptake, identification of this putative target of arsenite within the GLUT4-storage compartment may indicate whether it is a target for future strategies in prevention of diabetic cardiomyopathy.
Assuntos
Arsenitos/farmacologia , Antígenos CD36/metabolismo , Transportador de Glucose Tipo 4/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , Miócitos Cardíacos/efeitos dos fármacos , Animais , MAP Quinases Reguladas por Sinal Extracelular/fisiologia , Ácidos Graxos/metabolismo , Glucose/metabolismo , Masculino , Miócitos Cardíacos/metabolismo , Transporte Proteico/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/fisiologia , Ratos , Ratos Endogâmicos Lew , Proteínas Quinases S6 Ribossômicas/fisiologia , Proteínas Quinases p38 Ativadas por Mitógeno/fisiologiaRESUMO
Contraction of rat cardiac myocytes induces translocation of fatty acid translocase (FAT)/CD36 and GLUT4 from intracellular stores to the sarcolemma, leading to enhanced rates of long-chain fatty acid (FA) and glucose uptake, respectively. Because intracellular AMP/ATP is elevated in contracting cardiac myocytes, we investigated whether activation of AMP-activated protein kinase (AMP kinase) is involved in contraction-inducible FAT/CD36 translocation. The cell-permeable adenosine analog 5-aminoimidazole-4-carboxamide-1-beta-D-ribofuranoside (AICAR) and the mitochondrial inhibitor oligomycin, similar to 4-Hz electrostimulation, evoked a more than threefold activation of cardiomyocytic AMP kinase. Both AICAR and oligomycin stimulated FA uptake into noncontracting myocytes by 1.4- and 2.0-fold, respectively, but were ineffective in 4 Hz-contracting myocytes. These findings indicate that both agents stimulate FA uptake by a similar mechanism as electrostimulation, involving activation of AMP kinase, as evidenced from phosphorylation of acetyl-CoA carboxylase. Furthermore, the stimulating effects of both AICAR and oligomycin were antagonized by blocking FAT/CD36 with sulfo-N-succinimidylpalmitate, but not by inhibiting phosphatidylinositol 3-kinase with wortmannin, indicating the involvement of FAT/CD36, but excluding a role for insulin signaling. Subcellular fractionation showed that oligomycin was able to mobilize intracellularly stored FAT/CD36 to the sarcolemma. We conclude that AMP kinase regulates cardiac FA use through mobilization of FAT/CD36 from a contraction-inducible intracellular storage compartment.
Assuntos
Aminoimidazol Carboxamida/análogos & derivados , Coração/fisiologia , Glicoproteínas de Membrana/metabolismo , Complexos Multienzimáticos/metabolismo , Contração Miocárdica/fisiologia , Transportadores de Ânions Orgânicos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Quinases Ativadas por AMP , Trifosfato de Adenosina/metabolismo , Aminoimidazol Carboxamida/farmacologia , Animais , Antígenos CD36/metabolismo , Desoxiglucose/metabolismo , Estimulação Elétrica , Ácidos Graxos não Esterificados/metabolismo , Coração/efeitos dos fármacos , Técnicas In Vitro , Insulina/farmacologia , Cinética , Masculino , Mitocôndrias Cardíacas/metabolismo , Miocárdio/enzimologia , Miocárdio/metabolismo , Oligomicinas/farmacologia , Ratos , Ratos Endogâmicos Lew , Ribonucleotídeos/farmacologia , Transdução de SinaisRESUMO
In obesity, the development of cardiomyopathy is associated with the accumulation of myocardial triacylglycerols (TAGs), possibly stemming from elevation of myocardial long-chain fatty acid (LCFA) uptake. Because LCFA uptake is regulated by insulin and contractions, we examined in cardiac myocytes from lean and obese Zucker rats the effects of insulin and the contraction-mimetic agent oligomycin on the initial rate of LCFA uptake, subcellular distribution of FAT/CD36, and LCFA metabolism. In cardiac myocytes from obese Zucker rats, under basal conditions, FAT/CD36 was relocated to the sarcolemma at the expense of intracellular stores. In addition, the LCFA uptake rate, LCFA esterification rate into TAGs, and the intracellular unesterified LCFA concentration each were significantly increased. All these metabolic processes were normalized by the FAT/CD36 inhibitor sulfo-N-succinimidyloleate, indicating its antidiabetic potential. In cardiac myocytes isolated from lean rats, in vitro administration of insulin induced the translocation of FAT/CD36 to the sarcolemma and stimulated initial rates of LCFA uptake and TAG esterification. In contrast, in myocytes from obese rats, insulin failed to alter the subcellular localization of FAT/CD36 and the rates of LCFA uptake and TAG esterification. In cardiac myocytes from lean and obese animals, oligomycin stimulated the initial rates of LCFA uptake and oxidation, although oligomycin only induced the translocation of FAT/CD36 to the sarcolemma in lean rats. The present results indicate that in cardiac myocytes from obese Zucker rats, a permanent relocation of FAT/CD36 to the sarcolemma is responsible for myocardial TAG accumulation. Furthermore, in vitro these cardiac myocytes, although sensitive to contraction-like stimulation, were completely insensitive to insulin, as the basal conditions in hyperinsulinemic, obese animals resemble the insulin-stimulated condition in lean littermates.