RESUMEN
Three transduction pathways are involved in amino acid (AA) sensing in liver: mammalian target of rapamycin (mTOR), AMP-activated protein kinase (AMPK), and general control nondepressible kinase 2 (GCN2). However, no study has investigated the involvement of these signaling pathways in hepatic AA sensing. To address the question of liver AA sensing and signaling in response to a high-protein (HP) dietary supply, we investigated the changes in the phosphorylation state of hepatic mTOR (p-mTOR), AMPKalpha (p-AMPKalpha), and GCN2 (p-GCN2) by Western blotting. In rats fed a HP diet for 14 days, the hepatic p-AMPKalpha and p-GCN2 were lower (P < 0.001), and those of both the p-mTOR and eukaryotic initiation factor 4E-binding protein-1 phosphorylation (p-4E-BP1) were higher (P < 0.01) compared with rats receiving a normal protein (NP) diet. In hepatocytes in primary culture, high AA concentration decreased AMPKalpha phosphorylation whether insulin was present or not (P < 0.01). Either AAs or insulin can stimulate p-mTOR, but this is not sufficient for 4E-BP1 phosphorylation that requires both (P < 0.01). As expected, branched-chain AAs (BCAA) or leucine stimulated the phosphorylation of mTOR, but both insulin and BCAA or leucine are required for 4E-BP1 phosphorylation. GCN2 phosphorylation was reduced by both AAs and insulin(P < 0.01), suggesting for the first time that the translation inhibitor GCN2 senses not only the AA deficiency but also the AA increase in the liver. The present findings demonstrate that AAs and insulin exert a coordinated action on translation and involved mTOR, AMPK, and GCN2 transduction pathways.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Aminoácidos/metabolismo , Proteínas en la Dieta/metabolismo , Metabolismo Energético/fisiología , Hígado/metabolismo , Proteínas Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Animales , Western Blotting , Supervivencia Celular/fisiología , Proteínas en la Dieta/administración & dosificación , Glucosa/metabolismo , Hepatocitos/enzimología , Hepatocitos/metabolismo , Insulina/metabolismo , Hígado/enzimología , Masculino , Fosforilación , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/genética , ARN/química , ARN/genética , Ratas , Ratas Wistar , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Serina-Treonina Quinasas TORRESUMEN
Type 2 (non-insulin-dependent) diabetes mellitus is a progressive metabolic disorder arising from genetic and environmental factors that impair beta cell function and insulin action in peripheral tissues. We identified reduced diacylglycerol kinase delta (DGKdelta) expression and DGK activity in skeletal muscle from type 2 diabetic patients. In diabetic animals, reduced DGKdelta protein and DGK kinase activity were restored upon correction of glycemia. DGKdelta haploinsufficiency increased diacylglycerol content, reduced peripheral insulin sensitivity, insulin signaling, and glucose transport, and led to age-dependent obesity. Metabolic flexibility, evident by the transition between lipid and carbohydrate utilization during fasted and fed conditions, was impaired in DGKdelta haploinsufficient mice. We reveal a previously unrecognized role for DGKdelta in contributing to hyperglycemia-induced peripheral insulin resistance and thereby exacerbating the severity of type 2 diabetes. DGKdelta deficiency causes peripheral insulin resistance and metabolic inflexibility. These defects in glucose and energy homeostasis contribute to mild obesity later in life.
Asunto(s)
Diabetes Mellitus Tipo 2/genética , Diacilglicerol Quinasa/metabolismo , Regulación hacia Abajo , Resistencia a la Insulina , Adulto , Envejecimiento , Animales , Diacilglicerol Quinasa/genética , Diglicéridos/metabolismo , Metabolismo Energético , Perfilación de la Expresión Génica , Glucosa/metabolismo , Humanos , Hiperglucemia/metabolismo , Metabolismo de los Lípidos , Masculino , Ratones , Músculo Esquelético/metabolismo , Obesidad , Proteína Quinasa C/metabolismo , Ratas , Ratas Wistar , Transducción de SeñalRESUMEN
5'-AMP-activated protein kinase (AMPK) is important for metabolic sensing. We used AMPKgamma3 mutant-overexpressing Tg-Prkag3(225Q) and AMPKgamma3-knockout Prkag3-/- mice to determine the role of the AMPKgamma3 isoform in exercise-induced metabolic and gene regulatory responses in skeletal muscle. Mice were studied after 2 h swimming or 2.5 h recovery. Exercise increased basal and insulin-stimulated glucose transport, with similar responses among genotypes. In Tg-Prkag3(225Q) mice, acetyl-CoA carboxylase (ACC) phosphorylation was increased and triglyceride content was reduced after exercise, suggesting that this mutation promotes greater reliance on lipid oxidation. In contrast, ACC phosphorylation and triglyceride content was similar between wild-type and Prkag3-/- mice. Expression of genes involved in lipid and glucose metabolism was altered by genetic modification of AMPKgamma3. Expression of lipoprotein lipase 1, carnitine palmitoyl transferase 1b, and 3-hydroxyacyl-CoA dehydrogenase was increased in Tg-Prkag3(225Q) mice, with opposing effects in Prkag3-/- mice after exercise. GLUT4, hexokinase II (HKII), and glycogen synthase mRNA expression was increased in Tg-Prkag3(225Q) mice after exercise. GLUT4 and HKII mRNA expression was increased in wild-type mice and blunted in Prkag3-/- mice after recovery. In conclusion, the Prkag3(225Q) mutation, rather than presence of a functional AMPKgamma3 isoform, directly promotes metabolic and gene regulatory responses along lipid oxidative pathways in skeletal muscle after endurance exercise.
Asunto(s)
Proteínas Quinasas/deficiencia , Proteínas Quinasas/metabolismo , Proteínas Quinasas Activadas por AMP , Sustitución de Aminoácidos , Animales , Glucemia/efectos de los fármacos , Glucemia/metabolismo , Glucosa/metabolismo , Humanos , Insulina/farmacología , Ratones , Ratones Noqueados , Ratones Transgénicos , Músculo Esquelético/enzimología , Músculo Esquelético/fisiología , Condicionamiento Físico Animal , Natación , Triglicéridos/metabolismoRESUMEN
5'-AMP-activated protein kinase (AMPK) is a metabolic stress sensor present in all eukaryotes. A dominant missense mutation (R225Q) in pig PRKAG3, encoding the muscle-specific gamma3 isoform, causes a marked increase in glycogen content. To determine the functional role of the AMPK gamma3 isoform, we generated transgenic mice with skeletal muscle-specific expression of wild type or mutant (225Q) mouse gamma3 as well as Prkag3 knockout mice. Glycogen resynthesis after exercise was impaired in AMPK gamma3 knock-out mice and markedly enhanced in transgenic mutant mice. An AMPK activator failed to increase skeletal muscle glucose uptake in AMPK gamma3 knock-out mice, whereas contraction effects were preserved. When placed on a high fat diet, transgenic mutant mice but not knock-out mice were protected against excessive triglyceride accumulation and insulin resistance in skeletal muscle. Transfection experiments reveal the R225Q mutation is associated with higher basal AMPK activity and diminished AMP dependence. Our results validate the muscle-specific AMPK gamma3 isoform as a therapeutic target for prevention and treatment of insulin resistance.
Asunto(s)
Metabolismo de los Lípidos , Complejos Multienzimáticos/química , Complejos Multienzimáticos/genética , Músculo Esquelético/metabolismo , Proteínas Quinasas/química , Proteínas Quinasas/genética , Proteínas Serina-Treonina Quinasas/química , Proteínas Serina-Treonina Quinasas/genética , Proteínas Quinasas Activadas por AMP , Animales , Glucemia/metabolismo , Células COS , ADN Complementario/metabolismo , Glucosa/metabolismo , Glucógeno/metabolismo , Glucólisis , Insulina/metabolismo , Ratones , Ratones Noqueados , Ratones Transgénicos , Modelos Genéticos , Mutación Missense , Isoformas de Proteínas , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Porcinos , Temperatura , Transfección , Triglicéridos/metabolismoRESUMEN
Effects of diverse stimuli, including insulin, muscle contraction, and phorbol 12-myristate-13-acetate (PMA), were determined on phosphorylation of mitogen-activated protein kinase (MAPK) signaling modules (c-Jun NH(2)-terminal kinase [JNK], p38 MAPK, and extracellular signal-related kinase [ERK1/2]) in skeletal muscle from lean and ob/ob mice. Insulin increased phosphorylation of JNK, p38 MAPK, and ERK1/2 in isolated extensor digitorum longus (EDL) and soleus muscle from lean mice in a time- and dose-dependent manner. Muscle contraction and PMA also elicited robust effects on these parallel MAPK modules. Insulin action on JNK, p38 MAPK, and ERK1/2 phosphorylation was significantly impaired in EDL and soleus muscle from ob/ob mice. In contrast, muscle contraction-mediated JNK, p38 MAPK, and ERK1/2 phosphorylation was preserved. PMA effects on phosphorylation of JNK and ERK1/2 were normal in ob/ob mice, whereas effects on p38 MAPK were abolished. In conclusion, insulin, contraction, and PMA activate MAPK signaling in skeletal muscle. Insulin-mediated responses on MAPK signaling are impaired in skeletal muscle from ob/ob mice, whereas the effect of contraction is generally well preserved. In addition, PMA-induced phosphorylation of JNK and ERK1/2 are preserved, whereas p38 MAPK pathways are impaired in skeletal muscle from ob/ob mice. Thus, appropriate MAPK responses can be elicited in insulin-resistant skeletal muscle via an insulin-independent mechanism.
Asunto(s)
Insulina/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Contracción Muscular , Obesidad/fisiopatología , Acetato de Tetradecanoilforbol/farmacología , Animales , Femenino , Proteínas Quinasas JNK Activadas por Mitógenos , Ratones , Ratones Endogámicos C57BL , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Obesidad/genética , Obesidad/metabolismo , Fosforilación/efectos de los fármacos , Factores de Tiempo , Proteínas Quinasas p38 Activadas por MitógenosRESUMEN
We determined basal and insulin-stimulated responses on signaling intermediates in soleus skeletal muscle from male Wistar and diabetic Goto-Kakizaki (GK) rats. Rats were infused with glucose (5 or 20 mm) for 3 h, followed by a continuous infusion of saline or insulin (3 U/kg.h) for 20 min. Under euglycemic and hyperglycemic conditions, basal and insulin-stimulated action on phosphatidylinositol (PI) 3-kinase, protein kinase B/Akt, and ERK were reduced in GK rats, whereas insulin-stimulated protein kinase C (PKC)zeta activity was not altered. Interestingly, basal PKCzeta activity was increased under hyperglycemic conditions in GK and Wistar rats. This finding of increased PKCzeta activity was confirmed in vitro in isolated soleus muscle exposed to high extracellular glucose, and occurred concomitant with an increase in PI-dependent kinase 1 (PDK-1) activity. The glucose effects were not specific to PKCzeta, because an increase in phosphorylation of PKCalpha/beta and PKCdelta, but not PKCtheta, in isolated soleus muscle exposed to 25 mm glucose was observed. In conclusion, insulin signaling defects in diabetic GK rats are not corrected by an acute normalization of glycemia. Interestingly, acute hyperglycemia leads to a parallel increase in PDK-1, PKCalpha/beta, PKCdelta, and PKCzeta phosphorylation/activity via a PI 3-kinase-protein kinase B/Akt-independent mechanism. The long-term consequence of elevated PDK-1 and PKC phosphorylation/activity should be considered in the context of diabetes mellitus, as hyperglycemia is a clinical feature of this disease.
Asunto(s)
Diabetes Mellitus Experimental/metabolismo , Hiperglucemia/metabolismo , Músculo Esquelético/metabolismo , Transducción de Señal/fisiología , Proteínas Quinasas Dependientes de 3-Fosfoinosítido , Animales , Activación Enzimática/efectos de los fármacos , Glucosa/farmacología , Insulina/metabolismo , Isoenzimas/metabolismo , Masculino , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Fosfotirosina/metabolismo , Proteína Quinasa C/metabolismo , Proteína Quinasa C beta , Proteína Quinasa C-alfa , Proteína Quinasa C-delta , Proteína Quinasa C-theta , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Proto-Oncogénicas c-akt , Ratas , Ratas Mutantes , Ratas Wistar , Transducción de Señal/efectos de los fármacosRESUMEN
Glucose transport can be activated in skeletal muscle in response to insulin via activation of phosphoinositide (PI) 3-kinase and in response to contractions or hypoxia, presumably via activation of 5' AMP-activated protein kinase (AMPK). We determined the effects of insulin and muscle contraction/hypoxia on PI 3-kinase, AMPK, and glucose transport activity in epitrochlearis skeletal muscle from insulin-resistant Zucker (fa/ fa) rats. Insulin-stimulated glucose transport in isolated skeletal muscle was reduced 47% in obese versus lean rats, with a parallel 42% reduction in tyrosine-associated PI 3-kinase activity. Contraction and hypoxia elicited normal responses for glucose transport in skeletal muscle from insulin-resistant obese rats. Isoform-specific AMPK activity was measured in skeletal muscle in response to insulin, contraction, or hypoxia. Contraction increased AMPKalpha1 activity 2.3-fold in lean rats, whereas no effect was noted in obese rats. Hypoxia increased AMPKalpha1 activity to a similar extent (more than sixfold) in lean and obese rats. Regardless of genotype, contraction, and hypoxia, each increased AMPKalpha2 activity more than fivefold, whereas insulin did not alter either AMPKalpha1 or -alpha2 activity in skeletal muscle. In conclusion, obesity-related insulin resistance is associated with an isoform-specific impairment in AMPKalpha1 in response to contraction. However, this impairment does not appear to affect contraction-stimulated glucose transport. Activation of AMPKalpha2 in response to muscle contraction/ exercise is associated with a parallel and normal increase in glucose transport in insulin-resistant skeletal muscle.