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Non-classical gluconeogenesis-dependent glucose metabolism in Rhipicephalus microplus embryonic cell line BME26.

da Silva RM, Noce BD, Waltero CF, Costa EP, de Abreu LA, Githaka NW, Moraes J, Gomes HF, Konnai S, Vaz Ida S, Ohashi K, Logullo C - Int J Mol Sci (2015)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Chemistry and Function of Proteins and Peptides, Animal Experimentation Unit, UENF, Av. Alberto Lamego, 2000, Horto, CEP 28013-602 Campos dos Goytacazes, RJ, Brazil. rjrenato@ig.com.br.

ABSTRACT
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.

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Gluconeogenic enzymes have transcriptional control by GSK3 in BME26 silenced-cells. Transcriptional analysis of PEPCK (A) and glucose-6 phosphatase (B), gluconeogenic key-enzymes, in embryonic Rhipicephalus microplus cells (BME26) in response to GSK3 silencing (C). Control: cells maintained with 50 mM of glucose; Low: cell maintained without glucose addition; and High: cells maintained with 100 mM of glucose. The experiment was performed with three independent biological samples in three experimental replicates each (**p < 0.001, paired T test).
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ijms-16-01821-f008: Gluconeogenic enzymes have transcriptional control by GSK3 in BME26 silenced-cells. Transcriptional analysis of PEPCK (A) and glucose-6 phosphatase (B), gluconeogenic key-enzymes, in embryonic Rhipicephalus microplus cells (BME26) in response to GSK3 silencing (C). Control: cells maintained with 50 mM of glucose; Low: cell maintained without glucose addition; and High: cells maintained with 100 mM of glucose. The experiment was performed with three independent biological samples in three experimental replicates each (**p < 0.001, paired T test).

Mentions: These results demonstrate a differential genetic regulation between cell culture and in vivo models. Nevertheless, both gluconeogenesis enzymes were regulated in a coordinated manner, with similar transcriptional profiles. Our group has been investigating the role of GSK3 in a wide range of metabolic processes, including gluconeogenesis. GSK3 knockdown induces increases in PEPCK and GP transcription levels (Figure 8), suggesting an indirect genetic regulation of the gluconeogenesis pathway through GSK3. A high silencing rate of essential metabolic genes usually leads to cell lethality. Specifically, a GSK3 knockdown (around 90%) in female mosquito Aedes fluviatilis prevented them from developing their ovaries as well as egg laying. So, in a new experiment, GSK3 transcription was reduced by 30% to evaluate the effect of GSK3 inhibition on insect embryogenesis [44]. Similarly, we induced a higher efficiency in GSK3 silencing in the BME26 cells previously, leading to cell death (data not shown). Thus, a methodology was adopted to interfere in the transcriptional response, but without inducing cell death. At enzymatic level, GSK3 is regulated by phosphorylation, and a 15% of suppression may not affect the metabolism overall substantially. Effectively, the low rate in GSK3 silencing afforded to analyze the effect of GSK3 reductions in the transcription of gluconeogenesis genes.


Non-classical gluconeogenesis-dependent glucose metabolism in Rhipicephalus microplus embryonic cell line BME26.

da Silva RM, Noce BD, Waltero CF, Costa EP, de Abreu LA, Githaka NW, Moraes J, Gomes HF, Konnai S, Vaz Ida S, Ohashi K, Logullo C - Int J Mol Sci (2015)

Gluconeogenic enzymes have transcriptional control by GSK3 in BME26 silenced-cells. Transcriptional analysis of PEPCK (A) and glucose-6 phosphatase (B), gluconeogenic key-enzymes, in embryonic Rhipicephalus microplus cells (BME26) in response to GSK3 silencing (C). Control: cells maintained with 50 mM of glucose; Low: cell maintained without glucose addition; and High: cells maintained with 100 mM of glucose. The experiment was performed with three independent biological samples in three experimental replicates each (**p < 0.001, paired T test).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4307336&req=5

ijms-16-01821-f008: Gluconeogenic enzymes have transcriptional control by GSK3 in BME26 silenced-cells. Transcriptional analysis of PEPCK (A) and glucose-6 phosphatase (B), gluconeogenic key-enzymes, in embryonic Rhipicephalus microplus cells (BME26) in response to GSK3 silencing (C). Control: cells maintained with 50 mM of glucose; Low: cell maintained without glucose addition; and High: cells maintained with 100 mM of glucose. The experiment was performed with three independent biological samples in three experimental replicates each (**p < 0.001, paired T test).
Mentions: These results demonstrate a differential genetic regulation between cell culture and in vivo models. Nevertheless, both gluconeogenesis enzymes were regulated in a coordinated manner, with similar transcriptional profiles. Our group has been investigating the role of GSK3 in a wide range of metabolic processes, including gluconeogenesis. GSK3 knockdown induces increases in PEPCK and GP transcription levels (Figure 8), suggesting an indirect genetic regulation of the gluconeogenesis pathway through GSK3. A high silencing rate of essential metabolic genes usually leads to cell lethality. Specifically, a GSK3 knockdown (around 90%) in female mosquito Aedes fluviatilis prevented them from developing their ovaries as well as egg laying. So, in a new experiment, GSK3 transcription was reduced by 30% to evaluate the effect of GSK3 inhibition on insect embryogenesis [44]. Similarly, we induced a higher efficiency in GSK3 silencing in the BME26 cells previously, leading to cell death (data not shown). Thus, a methodology was adopted to interfere in the transcriptional response, but without inducing cell death. At enzymatic level, GSK3 is regulated by phosphorylation, and a 15% of suppression may not affect the metabolism overall substantially. Effectively, the low rate in GSK3 silencing afforded to analyze the effect of GSK3 reductions in the transcription of gluconeogenesis genes.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Chemistry and Function of Proteins and Peptides, Animal Experimentation Unit, UENF, Av. Alberto Lamego, 2000, Horto, CEP 28013-602 Campos dos Goytacazes, RJ, Brazil. rjrenato@ig.com.br.

ABSTRACT
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.

Show MeSH