Limits...
HIF-1 and c-Src mediate increased glucose uptake induced by endothelin-1 and connexin43 in astrocytes.

Valle-Casuso JC, González-Sánchez A, Medina JM, Tabernero A - PLoS ONE (2012)

Bottom Line: Indeed, our results show that silencing Cx43 increased HIF-1α and reduced the effect of ET-1 on HIF-1α, indicating that the effect of ET-1 on HIF-1α is mediated by Cx43.In addition, when c-Src activity was inhibited neither ET-1 nor silencing Cx43 were able to up-regulate HIF-1α.In conclusion, our results suggest that ET-1 by down-regulating Cx43 activates c-Src, which in turn increases HIF-1α leading to the up-regulation of the machinery required to take up glucose in astrocytes.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica y Biología Molecular, Instituto de Neurociencias de Castilla y León (INCYL), Universidad de Salamanca, Salamanca, Spain.

ABSTRACT
In previous work we showed that endothelin-1 (ET-1) increases the rate of glucose uptake in astrocytes, an important aspect of brain function since glucose taken up by astrocytes is used to supply the neurons with metabolic substrates. In the present work we sought to identify the signalling pathway responsible for this process in primary culture of rat astrocytes. Our results show that ET-1 promoted an increase in the transcription factor hypoxia-inducible factor-1α (HIF-1α) in astrocytes, as shown in other cell types. Furthermore, HIF-1α-siRNA experiments revealed that HIF-1α participates in the effects of ET-1 on glucose uptake and on the expression of GLUT-1, GLUT-3, type I and type II hexokinase. We previously reported that these effects of ET-1 are mediated by connexin43 (Cx43), the major gap junction protein in astrocytes. Indeed, our results show that silencing Cx43 increased HIF-1α and reduced the effect of ET-1 on HIF-1α, indicating that the effect of ET-1 on HIF-1α is mediated by Cx43. The activity of oncogenes such as c-Src can up-regulate HIF-1α. Since Cx43 interacts with c-Src, we investigated the participation of c-Src in this pathway. Interestingly, both the treatment with ET-1 and with Cx43-siRNA increased c-Src activity. In addition, when c-Src activity was inhibited neither ET-1 nor silencing Cx43 were able to up-regulate HIF-1α. In conclusion, our results suggest that ET-1 by down-regulating Cx43 activates c-Src, which in turn increases HIF-1α leading to the up-regulation of the machinery required to take up glucose in astrocytes. Cx43 expression can be reduced in response not only to ET-1 but also to various physiological and pathological stimuli. This study contributes to the identification of the signalling pathway evoked after Cx43 down-regulation that results in increased glucose uptake in astrocytes. Interestingly, this is the first evidence linking Cx43 to HIF-1, which is a master regulator of glucose metabolism.

Show MeSH

Related in: MedlinePlus

Effect of ET-1 on the expression of HIF-1α in astrocytes.Astrocytes were incubated in the absence (control) or presence of 0.1 µM ET-1 for the indicated times. Then, the expression of HIF-1α and Cx43 was analysed by Western blot. A) Representative Western blot of HIF-1α, Cx43 and GAPDH showing that ET-1 up-regulated HIF-1α and down-regulated Cx43. B) HIF-1α quantification. C) Cx43 quantification. The results are expressed as percentages of the level found in the controls at time 0. ***p<0.001 versus the corresponding controls.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3285680&req=5

pone-0032448-g001: Effect of ET-1 on the expression of HIF-1α in astrocytes.Astrocytes were incubated in the absence (control) or presence of 0.1 µM ET-1 for the indicated times. Then, the expression of HIF-1α and Cx43 was analysed by Western blot. A) Representative Western blot of HIF-1α, Cx43 and GAPDH showing that ET-1 up-regulated HIF-1α and down-regulated Cx43. B) HIF-1α quantification. C) Cx43 quantification. The results are expressed as percentages of the level found in the controls at time 0. ***p<0.001 versus the corresponding controls.

Mentions: In previous work we showed that ET-1 increased the rate of glucose uptake in astrocytes. Thus, ET-1 up-regulated GLUT-1 and Hx-1 and induced the expression of isoforms not normally expressed in astrocytes, such as GLUT-3 and Hx-2 [18], [26]. Since these proteins are target genes of HIF-1α, in this work we investigated whether the treatment with ET-1 modified HIF-1α levels in astrocytes, as it has been shown in other cell types [28], [29], [30]. Our results show that the treatment with 0.1 µM ET-1 strongly increased (by about 150%) the levels of HIF-1α (Figures 1A and B). This effect was evident after two hours of treatment and persisted for at least 48 h.


HIF-1 and c-Src mediate increased glucose uptake induced by endothelin-1 and connexin43 in astrocytes.

Valle-Casuso JC, González-Sánchez A, Medina JM, Tabernero A - PLoS ONE (2012)

Effect of ET-1 on the expression of HIF-1α in astrocytes.Astrocytes were incubated in the absence (control) or presence of 0.1 µM ET-1 for the indicated times. Then, the expression of HIF-1α and Cx43 was analysed by Western blot. A) Representative Western blot of HIF-1α, Cx43 and GAPDH showing that ET-1 up-regulated HIF-1α and down-regulated Cx43. B) HIF-1α quantification. C) Cx43 quantification. The results are expressed as percentages of the level found in the controls at time 0. ***p<0.001 versus the corresponding controls.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3285680&req=5

pone-0032448-g001: Effect of ET-1 on the expression of HIF-1α in astrocytes.Astrocytes were incubated in the absence (control) or presence of 0.1 µM ET-1 for the indicated times. Then, the expression of HIF-1α and Cx43 was analysed by Western blot. A) Representative Western blot of HIF-1α, Cx43 and GAPDH showing that ET-1 up-regulated HIF-1α and down-regulated Cx43. B) HIF-1α quantification. C) Cx43 quantification. The results are expressed as percentages of the level found in the controls at time 0. ***p<0.001 versus the corresponding controls.
Mentions: In previous work we showed that ET-1 increased the rate of glucose uptake in astrocytes. Thus, ET-1 up-regulated GLUT-1 and Hx-1 and induced the expression of isoforms not normally expressed in astrocytes, such as GLUT-3 and Hx-2 [18], [26]. Since these proteins are target genes of HIF-1α, in this work we investigated whether the treatment with ET-1 modified HIF-1α levels in astrocytes, as it has been shown in other cell types [28], [29], [30]. Our results show that the treatment with 0.1 µM ET-1 strongly increased (by about 150%) the levels of HIF-1α (Figures 1A and B). This effect was evident after two hours of treatment and persisted for at least 48 h.

Bottom Line: Indeed, our results show that silencing Cx43 increased HIF-1α and reduced the effect of ET-1 on HIF-1α, indicating that the effect of ET-1 on HIF-1α is mediated by Cx43.In addition, when c-Src activity was inhibited neither ET-1 nor silencing Cx43 were able to up-regulate HIF-1α.In conclusion, our results suggest that ET-1 by down-regulating Cx43 activates c-Src, which in turn increases HIF-1α leading to the up-regulation of the machinery required to take up glucose in astrocytes.

View Article: PubMed Central - PubMed

Affiliation: Departamento de Bioquímica y Biología Molecular, Instituto de Neurociencias de Castilla y León (INCYL), Universidad de Salamanca, Salamanca, Spain.

ABSTRACT
In previous work we showed that endothelin-1 (ET-1) increases the rate of glucose uptake in astrocytes, an important aspect of brain function since glucose taken up by astrocytes is used to supply the neurons with metabolic substrates. In the present work we sought to identify the signalling pathway responsible for this process in primary culture of rat astrocytes. Our results show that ET-1 promoted an increase in the transcription factor hypoxia-inducible factor-1α (HIF-1α) in astrocytes, as shown in other cell types. Furthermore, HIF-1α-siRNA experiments revealed that HIF-1α participates in the effects of ET-1 on glucose uptake and on the expression of GLUT-1, GLUT-3, type I and type II hexokinase. We previously reported that these effects of ET-1 are mediated by connexin43 (Cx43), the major gap junction protein in astrocytes. Indeed, our results show that silencing Cx43 increased HIF-1α and reduced the effect of ET-1 on HIF-1α, indicating that the effect of ET-1 on HIF-1α is mediated by Cx43. The activity of oncogenes such as c-Src can up-regulate HIF-1α. Since Cx43 interacts with c-Src, we investigated the participation of c-Src in this pathway. Interestingly, both the treatment with ET-1 and with Cx43-siRNA increased c-Src activity. In addition, when c-Src activity was inhibited neither ET-1 nor silencing Cx43 were able to up-regulate HIF-1α. In conclusion, our results suggest that ET-1 by down-regulating Cx43 activates c-Src, which in turn increases HIF-1α leading to the up-regulation of the machinery required to take up glucose in astrocytes. Cx43 expression can be reduced in response not only to ET-1 but also to various physiological and pathological stimuli. This study contributes to the identification of the signalling pathway evoked after Cx43 down-regulation that results in increased glucose uptake in astrocytes. Interestingly, this is the first evidence linking Cx43 to HIF-1, which is a master regulator of glucose metabolism.

Show MeSH
Related in: MedlinePlus