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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 response increase in high glucose concentration in BME26 cells. Transcriptional analysis of PEPCK (A) and glucose-6-phosphatase (B), gluconeogenic key-enzymes, in embryonic Rhipicephalus microplus cells (BME26) in response to glucose treatment. 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, ANOVA).
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ijms-16-01821-f007: Gluconeogenic response increase in high glucose concentration in BME26 cells. Transcriptional analysis of PEPCK (A) and glucose-6-phosphatase (B), gluconeogenic key-enzymes, in embryonic Rhipicephalus microplus cells (BME26) in response to glucose treatment. 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, ANOVA).

Mentions: Gluconeogenesis produces glucose from non-glycosidic compounds, and it is an important strategy for the maintenance of cell energy homeostasis. PEPCK and GP are regulatory enzymes that catalyze the initial and final steps of gluconeogenesis, respectively [37,38]. GP removes a phosphate from glucose-6-phosphate to produce free glucose. Both enzymes exhibited similar transcriptional profiles across glucose treatments, showing reduced amounts of transcripts in the low glucose-treated cells (Figure 7). In the high-glucose cells, the opposite was observed, with increased transcription levels of both PEPCK and GP. Under normal physiological conditions, when glucose levels become low, as in starvation, the gluconeogenic flux accelerates [39,40]. In such cases, it is expected that these enzymes will undergo higher transcription when glucose levels are reduced, particularly PEPCK, as an enzyme that is mainly regulated by transcription in mammals. Surprisingly, in BME26 cells, this transcriptional profile was reversed. In cell culture, because there are no groups of specialized cells as seen in vivo, glucose dephosphorylation catalyzed by GP would result in the release of glucose and, consequently, the loss of this metabolite. Cells treated with low glucose may lack carbohydrate reserves [15], and a decrease in GP transcription is necessary to avoid the additional loss of glucose content. On the other hand, GP transcription increases in high-glucose cells. Massillon [41] observed an increase in GP transcription when glucose levels were elevated in hepatocyte cell culture, showing a dose-dependent response. The uptake of large amounts of glucose and the consequent phosphorylation of glucose inhibits HK activity as a result of elevated glucose-6-phosphate levels, leading to ROS generation by mitochondria [22]. In the context of cell culture, GP may be necessary to allow the diffusion of excess glucose away to avoid cell damage. PEPCK is also increased at the transcriptional level in high glucose-treated cells (Figure 7A). In addition to regulating gluconeogenesis, PEPCK regulates glyceroneogenesis, a pathway required for free fatty acid re-esterification to maintain an active level of triglyceride synthesis [38,42]. Under high carbohydrate availability, the flux through glyceroneogenesis increases [38]. Due to this phenomenon, we postulated that an increase in PEPCK transcription reflects a condition of high energy availability and possible glyceroneogenesis induction. Such genic profile in high glucose tick cells is very similar to observed in mammalian diabetic cells [43]. Interestingly, the glycolysis presented increased at this same moment, suggesting a deviation of pyruvate produced by glycolysis to gluconeogenesis or glyceroneogenesis (Figure 1). Indeed, a change in cell phenotype characterized by altered morphology was observed following glucose treatments. Cell culture heterogeneity was higher in the low-glucose cells than in control cells.


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 response increase in high glucose concentration in BME26 cells. Transcriptional analysis of PEPCK (A) and glucose-6-phosphatase (B), gluconeogenic key-enzymes, in embryonic Rhipicephalus microplus cells (BME26) in response to glucose treatment. 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, ANOVA).
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Related In: Results  -  Collection

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ijms-16-01821-f007: Gluconeogenic response increase in high glucose concentration in BME26 cells. Transcriptional analysis of PEPCK (A) and glucose-6-phosphatase (B), gluconeogenic key-enzymes, in embryonic Rhipicephalus microplus cells (BME26) in response to glucose treatment. 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, ANOVA).
Mentions: Gluconeogenesis produces glucose from non-glycosidic compounds, and it is an important strategy for the maintenance of cell energy homeostasis. PEPCK and GP are regulatory enzymes that catalyze the initial and final steps of gluconeogenesis, respectively [37,38]. GP removes a phosphate from glucose-6-phosphate to produce free glucose. Both enzymes exhibited similar transcriptional profiles across glucose treatments, showing reduced amounts of transcripts in the low glucose-treated cells (Figure 7). In the high-glucose cells, the opposite was observed, with increased transcription levels of both PEPCK and GP. Under normal physiological conditions, when glucose levels become low, as in starvation, the gluconeogenic flux accelerates [39,40]. In such cases, it is expected that these enzymes will undergo higher transcription when glucose levels are reduced, particularly PEPCK, as an enzyme that is mainly regulated by transcription in mammals. Surprisingly, in BME26 cells, this transcriptional profile was reversed. In cell culture, because there are no groups of specialized cells as seen in vivo, glucose dephosphorylation catalyzed by GP would result in the release of glucose and, consequently, the loss of this metabolite. Cells treated with low glucose may lack carbohydrate reserves [15], and a decrease in GP transcription is necessary to avoid the additional loss of glucose content. On the other hand, GP transcription increases in high-glucose cells. Massillon [41] observed an increase in GP transcription when glucose levels were elevated in hepatocyte cell culture, showing a dose-dependent response. The uptake of large amounts of glucose and the consequent phosphorylation of glucose inhibits HK activity as a result of elevated glucose-6-phosphate levels, leading to ROS generation by mitochondria [22]. In the context of cell culture, GP may be necessary to allow the diffusion of excess glucose away to avoid cell damage. PEPCK is also increased at the transcriptional level in high glucose-treated cells (Figure 7A). In addition to regulating gluconeogenesis, PEPCK regulates glyceroneogenesis, a pathway required for free fatty acid re-esterification to maintain an active level of triglyceride synthesis [38,42]. Under high carbohydrate availability, the flux through glyceroneogenesis increases [38]. Due to this phenomenon, we postulated that an increase in PEPCK transcription reflects a condition of high energy availability and possible glyceroneogenesis induction. Such genic profile in high glucose tick cells is very similar to observed in mammalian diabetic cells [43]. Interestingly, the glycolysis presented increased at this same moment, suggesting a deviation of pyruvate produced by glycolysis to gluconeogenesis or glyceroneogenesis (Figure 1). Indeed, a change in cell phenotype characterized by altered morphology was observed following glucose treatments. Cell culture heterogeneity was higher in the low-glucose cells than in control cells.

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