Limits...
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.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.

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

Related in: MedlinePlus

Membrane integrity is unaffected in BME26 cells after glucose treatment. The cells were directly stained by adding Hoechst 33342 and propidium iodide. Glass slides were observed in a fluorescence microscope (model Eclipse 80i, Nikon), and pictures were obtained at 400× magnification. Control: cells maintained with 50 mM of glucose (A); Low: cell maintained without glucose addition (B); and High: cells maintained with 100 mM of glucose (C). The arrows indicate the shape of low-glucose cells and the triangles indicate the rounded shape of high-glucose cells. Scale bar: 10 μm.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4307336&req=5

ijms-16-01821-f004: Membrane integrity is unaffected in BME26 cells after glucose treatment. The cells were directly stained by adding Hoechst 33342 and propidium iodide. Glass slides were observed in a fluorescence microscope (model Eclipse 80i, Nikon), and pictures were obtained at 400× magnification. Control: cells maintained with 50 mM of glucose (A); Low: cell maintained without glucose addition (B); and High: cells maintained with 100 mM of glucose (C). The arrows indicate the shape of low-glucose cells and the triangles indicate the rounded shape of high-glucose cells. Scale bar: 10 μm.

Mentions: A cell viability assay was performed for the three culture conditions (Figure 3). With low glucose treatment, cell viability was lower than with other treatments. However, cell viability was enhanced with treatment with a high glucose concentration, when compared to control (cells maintained with a usual glucose concentration). Mitochondrial hexokinase activity is critical for sustaining constant ADP steady-state cycling, which in turn reduces the membrane potential and consequently decreases mitochondrial ROS formation, as previously described in rat brain cells [22,23]. Thus, glucose supports an increase in HK activity, leading to oxidative stress protection and higher cell survival [23]. Furthermore, if glucose availability is extremely high, the mitochondrial hexokinase activity decreases when ADP is produced. In this case, ROS production leads to a decrease in cell viability [24]. In this study, an increase in glucose disposal under high glucose condition is likely to improve the cell’s energetic fitness, resulting in higher viability, unlike cells cultured in low glucose levels. Furthermore, the microscopy analysis with propidium iodide did not show loss of membrane integrity, in any treatment (Figure 4).


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)

Membrane integrity is unaffected in BME26 cells after glucose treatment. The cells were directly stained by adding Hoechst 33342 and propidium iodide. Glass slides were observed in a fluorescence microscope (model Eclipse 80i, Nikon), and pictures were obtained at 400× magnification. Control: cells maintained with 50 mM of glucose (A); Low: cell maintained without glucose addition (B); and High: cells maintained with 100 mM of glucose (C). The arrows indicate the shape of low-glucose cells and the triangles indicate the rounded shape of high-glucose cells. Scale bar: 10 μm.
© Copyright Policy
Related In: Results  -  Collection

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

ijms-16-01821-f004: Membrane integrity is unaffected in BME26 cells after glucose treatment. The cells were directly stained by adding Hoechst 33342 and propidium iodide. Glass slides were observed in a fluorescence microscope (model Eclipse 80i, Nikon), and pictures were obtained at 400× magnification. Control: cells maintained with 50 mM of glucose (A); Low: cell maintained without glucose addition (B); and High: cells maintained with 100 mM of glucose (C). The arrows indicate the shape of low-glucose cells and the triangles indicate the rounded shape of high-glucose cells. Scale bar: 10 μm.
Mentions: A cell viability assay was performed for the three culture conditions (Figure 3). With low glucose treatment, cell viability was lower than with other treatments. However, cell viability was enhanced with treatment with a high glucose concentration, when compared to control (cells maintained with a usual glucose concentration). Mitochondrial hexokinase activity is critical for sustaining constant ADP steady-state cycling, which in turn reduces the membrane potential and consequently decreases mitochondrial ROS formation, as previously described in rat brain cells [22,23]. Thus, glucose supports an increase in HK activity, leading to oxidative stress protection and higher cell survival [23]. Furthermore, if glucose availability is extremely high, the mitochondrial hexokinase activity decreases when ADP is produced. In this case, ROS production leads to a decrease in cell viability [24]. In this study, an increase in glucose disposal under high glucose condition is likely to improve the cell’s energetic fitness, resulting in higher viability, unlike cells cultured in low glucose levels. Furthermore, the microscopy analysis with propidium iodide did not show loss of membrane integrity, in any treatment (Figure 4).

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.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.

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
Related in: MedlinePlus