RESUMO
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
Contraction-induced glucose uptake is only partly mediated by AMPK activation. We examined whether the diacylglycerol-sensitive protein kinase D (PKD; also known as novel PKC isoform mu) is also involved in the regulation of glucose uptake in the contracting heart. As an experimental model, we used suspensions of cardiac myocytes, which were electrically stimulated to contract or treated with the contraction-mimicking agent oligomycin. Induction of contraction at 4 Hz in cardiac myocytes or treatment with 1 microM oligomycin enhanced (i) autophosphorylation of PKD at Ser916 by 5.1- and 3.8-fold, respectively, (ii) phosphorylation of PKD's downstream target cardiac-troponin-I (cTnI) by 2.9- and 2.1-fold, respectively, and (iii) enzymatic activity of immunoprecipitated PKD towards the substrate peptide syntide-2 each by 1.5-fold. Although AMPK was also activated under these same conditions, in vitro phosphorylation assays and studies with cardiac myocytes from AMPKalpha2(-/-) mice indicated that activation of PKD occurs independent of AMPK activation. CaMKKbeta, and the cardiac-specific PKC isoforms alpha, delta, and epsilon were excluded as upstream kinases for PKD in contraction signaling because none of these kinases were activated by oligomycin. Stimulation of glucose uptake and induction of GLUT4 translocation in cardiac myocytes by contraction and oligomycin each were sensitive to inhibition by the PKC/PKD inhibitors staurosporin and calphostin-C. Together, these data elude to a role of PKD in contraction-induced GLUT4 translocation. Finally, the combined actions of PKD on cTnI phosphorylation and on GLUT4 translocation would efficiently link accelerated contraction mechanics to increased energy production when the heart is forced to increase its contractile activity.
Assuntos
Transportador de Glucose Tipo 4/metabolismo , Glucose/metabolismo , Complexos Multienzimáticos/metabolismo , Contração Muscular , Miócitos Cardíacos/metabolismo , Proteína Quinase C/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Proteínas Quinases Ativadas por AMP , Animais , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Desoxiglucose/metabolismo , Estimulação Elétrica , Ativação Enzimática , Peptídeos e Proteínas de Sinalização Intercelular , Masculino , Camundongos , Camundongos Knockout , Complexos Multienzimáticos/deficiência , Complexos Multienzimáticos/genética , Contração Muscular/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Miócitos Cardíacos/enzimologia , Naftalenos/farmacologia , Oligomicinas/farmacologia , Peptídeos/metabolismo , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/deficiência , Proteínas Serina-Treonina Quinases/genética , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacos , Estaurosporina/farmacologia , Troponina I/metabolismoRESUMO
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
Familial combined hyperlipidemia (FCHL) shows many features of the metabolic syndrome. The strong genetic component makes it an excellent model to study the genetic background of metabolic syndrome and insulin resistance. Adipose tissue is believed to contribute to, or even underlie, the FCHL phenotype and is an interesting target tissue for gene expression studies. However, interpretation of adipose tissue gene expression experiments is complex since expression differences cannot only arise as a direct consequence of a genetic trait, but may also reflect an adaptation to metabolic influences at the cellular level. In the present study, we measured gene expression levels in cultured primary human preadipocytes from FCHL and control subjects. Since isolated preadipocytes were allowed to replicate for weeks under standardized conditions, the contribution of previous metabolic influences is rather small whereas genetic defects are preserved and expressed in vitro. The main finding was up-regulation of CD36/FAT in FCHL preadipocytes, confirmed in two independent groups of subjects, and a concomitant increase in CD36/FAT-mediated fatty acid uptake. CD36/FAT overexpression has previously been shown to be associated with other insulin-resistant states. The present data suggest that CD36/FAT overexpression in FCHL occurs very early in adipocyte differentiation and may be of genetic origin.