RESUMEN
Fish are constantly exposed to microorganisms in the aquatic environment, many of which are bacterial pathogens. Bacterial pathogens activate the innate immune response in fish involving the production of pro-inflammatory molecules that, in addition to their immune-related role, can affect non-immune tissues. In the present study, we aimed at investigating how inflammatory responses can affect metabolic homeostasis in the gilthead seabream (Sparus aurata), a teleost of considerable economic importance in Southern European countries. Specifically, we mimicked a bacterial infection by in vivo administration of lipopolysaccharide (LPS, 6 mg/kg body weight) and measured metabolic parameters in the blood and, importantly, the mRNA expression levels of the three isotypes of peroxisome proliferator activated receptors (PPARα, ß, and γ) in metabolically-relevant tissues in seabream. PPARs are nuclear receptors that are important for lipid and carbohydrate metabolism in mammals and that act as biological sensors of altered lipid metabolism. We show here that LPS-induced inflammatory responses result in the modulation of triglyceride plasma levels that are accompanied most notably by a decrease in the hepatic mRNA expression levels of PPARα, ß, and γ and by the up-regulation of PPARγ expression only in adipose tissue and the anterior intestine. In addition, LPS-induced inflammation results in an increase in the hepatic mRNA expression and protein activity levels of members of the mitogen-activated protein kinase (MAPK) family, known in mammals to regulate the transcription and activity of PPARs. Our results provide evidence for the involvement of PPARs in the metabolic response to inflammatory stimuli in seabream and offer insights into the molecular mechanisms underlying the redirection of metabolic activities under inflammatory conditions in vertebrates.
RESUMEN
The physiological consequences of the activation of the immune system in fish are not well understood. In particular, skeletal muscle, due to its essential role in locomotion and whole-animal energy homeostasis, is a potentially important target of inflammation. In this study, we have evaluated the in vivo effects of lipopolysaccharide (LPS) on the white and red skeletal muscle transcriptome of the gilthead seabream (Sparus aurata) by microarray analysis at 24 and 72 h after injection. In white muscle, the transcriptomic response was characterized by an up-regulation of genes involved in carbohydrate catabolism and protein synthesis at 24 h and a complete reversal of this pattern at 72 h. In red muscle, an up-regulation of genes involved in carbohydrate catabolism and protein synthesis was observed only at 72 h after LPS administration. Interestingly, both white and red muscles showed a similar consistent down-regulation of immune genes at 72 h post-injection. However, genes involved in muscle contraction showed a general up-regulation in response to LPS in both types of muscle. In summary, LPS administration causes muscle type-specific responses regarding the expression of genes involved in carbohydrate and protein metabolism and a common decreased expression of immune genes in skeletal muscle, concomitant with increased expression of genes for contractile elements. Our results evidence a robust and tissue-specific transcriptomic response of the skeletal muscle to an acute inflammatory challenge.
Asunto(s)
Enfermedades de los Peces/inmunología , Inflamación/veterinaria , Lipopolisacáridos/inmunología , Músculo Esquelético/metabolismo , Dorada , Transcriptoma/genética , Animales , Cartilla de ADN/genética , ADN Complementario/biosíntesis , Enfermedades de los Peces/genética , Inflamación/inmunología , Análisis por Micromatrices/veterinaria , Contracción Muscular/genética , Contracción Muscular/inmunología , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Factores de Tiempo , Transcriptoma/inmunologíaRESUMEN
AMPK, a master metabolic switch, mediates the observed increase of glucose uptake in locomotory muscle of mammals during exercise. AMPK is activated by changes in the intracellular AMP:ATP ratio when ATP consumption is stimulated by contractile activity but also by AICAR and metformin, compounds that increase glucose transport in mammalian muscle cells. However, the possible role of AMPK in the regulation of glucose metabolism in skeletal muscle has not been investigated in other vertebrates, including fish. In this study, we investigated the effects of AMPK activators on glucose uptake, AMPK activity, cell surface levels of trout GLUT4 and expression of GLUT1 and GLUT4 as well as the expression of enzymes regulating glucose disposal and PGC1α in trout myotubes derived from a primary muscle cell culture. We show that AICAR and metformin significantly stimulated glucose uptake (1.6 and 1.3 fold, respectively) and that Compound C completely abrogated the stimulatory effects of the AMPK activators on glucose uptake. The combination of insulin and AMPK activators did not result in additive nor synergistic effects on glucose uptake. Moreover, exposure of trout myotubes to AICAR and metformin resulted in an increase in AMPK activity (3.8 and 3 fold, respectively). We also provide evidence suggesting that stimulation of glucose uptake by AMPK activators in trout myotubes may take place, at least in part, by increasing the cell surface and mRNA levels of trout GLUT4. Finally, AICAR increased the mRNA levels of genes involved in glucose disposal (hexokinase, 6-phosphofructokinase, pyruvate kinase and citrate synthase) and mitochondrial biogenesis (PGC-1α) and did not affect glycogen content or glycogen synthase mRNA levels in trout myotubes. Therefore, we provide evidence, for the first time in non-mammalian vertebrates, suggesting a potentially important role of AMPK in stimulating glucose uptake and utilization in the skeletal muscle of fish.
Asunto(s)
Proteínas Quinasas Activadas por AMP/fisiología , Evolución Molecular , Peces/metabolismo , Glucosa/metabolismo , Músculo Esquelético/enzimología , Animales , Transportador de Glucosa de Tipo 4 , Insulina/farmacología , Metformina/farmacología , Fibras Musculares Esqueléticas , Músculo Esquelético/efectos de los fármacos , TruchaRESUMEN
The proinflammatory cytokine TNF-α is known to have a direct action on skeletal muscle in mammals. However, little is known regarding the potential effects of cytokines on nonimmune tissues, particularly in skeletal muscle, in fish. The aim of this study was to investigate the effects of recombinant trout TNF-α (rtTNF-α) on skeletal muscle carbohydrate metabolism in rainbow trout (Oncorhynchus mykiss). We used a primary cell culture of muscle cells from rainbow trout to show that rtTNF-α stimulates glucose uptake in myoblasts and myotubes at concentrations that do not affect the viability of the cells, requiring de novo protein synthesis as shown by the impairment of rtTNF-α-stimulated glucose uptake by cycloheximide. With the use of specific inhibitors, we show that rtTNF-α-stimulated glucose uptake is mediated by the p38MAPK, NF-κB, and JNK pathways. Additionally, we provide evidence that the stimulatory effects of rtTNF-α on glucose uptake in trout skeletal muscle cells may be caused, at least in part, by an increase in the amount of GLUT4 at the plasma membrane. Incubation of trout muscle cells with conditioned medium from LPS-stimulated trout macrophages, enriched in TNF-α, increased glucose uptake. Our results indicate that recombinant, as well as native trout TNF-α, directly stimulates glucose uptake in trout muscle cells and provide evidence, for the first time in nonmammalian vertebrates, for a potential regulatory role of TNF-α in skeletal muscle metabolism.
Asunto(s)
Desoxiglucosa/metabolismo , Proteínas de Peces/metabolismo , Músculo Esquelético/metabolismo , Oncorhynchus mykiss/metabolismo , Células Satélite del Músculo Esquelético/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Animales , Transporte Biológico , Membrana Celular/metabolismo , Células Cultivadas , Medios de Cultivo Condicionados/metabolismo , Transportador de Glucosa de Tipo 4/genética , Transportador de Glucosa de Tipo 4/metabolismo , Insulina/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Lipopolisacáridos/farmacología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/inmunología , FN-kappa B/antagonistas & inhibidores , FN-kappa B/metabolismo , Oncorhynchus mykiss/genética , Oncorhynchus mykiss/inmunología , Comunicación Paracrina , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de la Síntesis de la Proteína/farmacología , Ratas , Proteínas Recombinantes/metabolismo , Células Satélite del Músculo Esquelético/efectos de los fármacos , Células Satélite del Músculo Esquelético/inmunología , Transfección , Proteínas Quinasas p38 Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Insulin is an important factor for the maintenance of glucose homeostasis, enhancing glucose uptake in its target tissues in a process that has been conserved between fish and mammals. In fish skeletal muscle cells, like in mammals, insulin promotes GLUT4 translocation to the plasma membrane and, consequently, glucose uptake, but its role regulating the expression of glucose transporters in vitro has not been demonstrated to date. Thus, we investigated the expression of GLUT4 and GLUT1 throughout skeletal muscle cell differentiation and their regulation by insulin and IGF-I using a primary culture of trout muscle cells. GLUT4 expression gradually increased during the muscle cell differentiation process, whereas GLUT1 expression remained fairly constant. Insulin and IGF-I similarly increased the mRNA levels of GLUT4 in myoblasts and myotubes. On the other hand, IGF-I appeared to be more potent than insulin in stimulating GLUT1 expression, particularly at the myoblast stage. Therefore, this work provides the first demonstration in nonmammalian vertebrates that insulin and IGF-I may act directly on trout muscle cells to regulate the expression of GLUT4 and GLUT1.