Regulation of GLUT3 and glucose uptake by the cAMP signalling pathway in the breast cancer cell line ZR-75.

Increased glucose uptake as a principal energy source is a requirement for the continued survival of tumour cells. Facilitative glucose transporter-1 (GLUT1) and -3 (GLUT3) have been previously shown to be present and regulated in breast cancer cells and are associated with poor patient prognosis. In cancer cells, the cAMP secondary messenger pathway is known to potentiate described glucose transporter activators and regulate cell fate. However, no regulation of the glucose transporters in breast cancer cells by cAMP has previously been examined. Herein, we determined in the well-characterized breast cancer cell line ZR-75, if the cAMP analogue 8-br-cAMP was capable of regulating GLUT1 and GLUT3 expression and thus glucose uptake. We demonstrated that 8-br-cAMP transiently up-regulates GLUT3 mRNA levels. The use of actinomycin-D and the cloning of 1,200 bp upstream of the human GLUT3 promoter demonstrated that this regulation was transcriptional. Immunocytochemistry and Western blotting confirmed that the increase in mRNA was reflected by an increase in protein levels. No notable regulation of GLUT1 in the presence of 8-br-cAMP was detected. Finally, we determined using the non-metabolizable glucose analogue 2-DOG if this up-regulation in GLUT3 increased glucose uptake. We observed the presence of two uptake components, one corresponding to the Km of GLUT1/4 and the other to GLUT3. A doubling in the uptake velocity was observed only at the Km corresponding to GLUT3. In conclusion, we demonstrate and characterize for the first time, an up-regulation of GLUT3 mRNA, protein and glucose uptake by the cAMP pathway in breast cancer cells.

Glial glucokinase expression in adult and post-natal development of the hypothalamic region.

It has recently been proposed that hypothalamic glial cells sense glucose levels and release lactate as a signal to activate adjacent neurons. GK (glucokinase), the hexokinase involved in glucose sensing in pancreatic beta-cells, is also expressed in the hypothalamus. However, it has not been clearly determined if glial and/or neuronal cells express this protein. Interestingly, tanycytes, the glia that cover the ventricular walls of the hypothalamus, are in contact with CSF (cerebrospinal fluid), the capillaries of the arcuate nucleus and adjacent neurons; this would be expected for a system that can detect and communicate changes in glucose concentration. Here, we demonstrated by Western-blot analysis, QRT-PCR [quantitative RT-PCR (reverse transcription-PCR)] and in situ hybridization that GK is expressed in tanycytes. Confocal microscopy and immuno-ultrastructural analysis revealed that GK is localized in the nucleus and cytoplasm of beta1-tanycytes. Furthermore, GK expression increased in these cells during the second week of post-natal development. Based on this evidence, we propose that tanycytes mediate, at least in part, the mechanism by which the hypothalamus detects changes in glucose concentrations.