RESUMO
Reactive oxygen species (ROS) are natural byproducts of metabolism that have toxic effects well documented in mammals. In hematophagous arthropods, however, these processes are not largely understood. Here, we describe that Rhipicephalus microplus ticks and embryonic cell line (BME26) employ an adaptive metabolic compensation mechanism that confers tolerance to hydrogen peroxide (H2O2) at concentrations too high for others organisms. Tick survival and reproduction are not affected by H2O2 exposure, while BME26 cells morphology was only mildly altered by the treatment. Furthermore, H2O2-tolerant BME26 cells maintained their proliferative capacity unchanged. We evaluated several genes involved in gluconeogenesis, glycolysis, and pentose phosphate pathway, major pathways for carbohydrate catabolism and anabolism, describing a metabolic mechanism that explains such tolerance. Genetic and catalytic control of the genes and enzymes associated with these pathways are modulated by glucose uptake and energy resource availability. Transient increase in ROS levels, oxygen consumption, and ROS-scavenger enzymes, as well as decreased mitochondrial superoxide levels, were indicative of cell adaptation to high H2O2 exposure, and suggested a tolerance strategy developed by BME26 cells to cope with oxidative stress. Moreover, NADPH levels increased upon H2O2 challenge, and this phenomenon was sustained mainly by G6PDH activity. Interestingly, G6PDH knockdown in BME26 cells did not impair H2O2 tolerance, but generated an increase in NADP-ICDH transcription. In agreement with the hypothesis of a compensatory NADPH production in these cells, NADP-ICDH knockdown increased G6PDH relative transcript level. The present study unveils the first metabolic evidence of an adaptive mechanism to cope with high H2O2 exposure and maintain redox balance in ticks.
Assuntos
Metabolismo dos Carboidratos/fisiologia , Peróxido de Hidrogênio/toxicidade , Oxidantes/toxicidade , Estresse Oxidativo/fisiologia , Rhipicephalus/metabolismo , Adaptação Fisiológica , Animais , Carboidratos/química , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Resistência a Medicamentos , Tolerância a Medicamentos/fisiologia , Glucosefosfato Desidrogenase/genética , Glucosefosfato Desidrogenase/metabolismo , NADP/análise , OxirreduçãoRESUMO
In this work we evaluated several genes involved in gluconeogenesis, glycolysis and glycogen metabolism, the major pathways for carbohydrate catabolism and anabolism, in the BME26 Rhipicephalus microplus embryonic cell line. Genetic and catalytic control of the genes and enzymes associated with these pathways are modulated by alterations in energy resource availability (primarily glucose). BME26 cells in media were investigated using three different glucose concentrations, and changes in the transcription levels of target genes in response to carbohydrate utilization were assessed. The results indicate that several genes, such as glycogen synthase (GS), glycogen synthase kinase 3 (GSK3), phosphoenolpyruvate carboxykinase (PEPCK), and glucose-6 phosphatase (GP) displayed mutual regulation in response to glucose treatment. Surprisingly, the transcription of gluconeogenic enzymes was found to increase alongside that of glycolytic enzymes, especially pyruvate kinase, with high glucose treatment. In addition, RNAi data from this study revealed that the transcription of gluconeogenic genes in BME26 cells is controlled by GSK-3. Collectively, these results improve our understanding of how glucose metabolism is regulated at the genetic level in tick cells.
Assuntos
Gluconeogênese , Glucose/metabolismo , Rhipicephalus/metabolismo , Animais , Linhagem Celular , Regulação da Expressão Gênica , Glucose/genética , Rhipicephalus/citologia , Rhipicephalus/embriologia , Rhipicephalus/genéticaRESUMO
BACKGROUND: Tick embryogenesis is a metabolically intensive process developed under tightly controlled conditions and whose components are poorly understood. METHODS: In order to characterize the role of AKT (protein kinase B) in glycogen metabolism and cell viability, glycogen determination, identification and cloning of an AKT from Rhipicephalus microplus were carried out, in parallel with experiments using RNA interference (RNAi) and chemical inhibition. RESULTS: A decrease in glycogen content was observed when AKT was chemically inhibited by 10-DEBC treatment, while GSK3 inhibition by alsterpaullone had an opposing effect. RmAKT ORF is 1584-bp long and encodes a polypeptide chain of 60.1 kDa. Phylogenetic and sequence analyses showed significant differences between vertebrate and tick AKTs. Either AKT or GSK3 knocked down cells showed a 70% reduction in target transcript levels, but decrease in AKT also reduced glycogen content, cell viability and altered cell membrane permeability. However, the GSK3 reduction promoted an increase in glycogen content. Additionally, either GSK3 inhibition or gene silencing had a protective effect on BME26 viability after exposure to ultraviolet radiation. R. microplus AKT and GSK3 were widely expressed during embryo development. Taken together, our data support an antagonistic role for AKT and GSK3, and strongly suggest that such a signaling axis is conserved in tick embryos, with AKT located upstream of GSK3. GENERAL SIGNIFICANCE: The AKT/GSK3 axis is conserved in tick in a way that integrates glycogen metabolism and cell survival, and exhibits phylogenic differences that could be important for the development of novel control methods.