Expression Glucose Research Articles

Elevated expression of glucose transporter‐1 in hypothalamic ependymal cells not involved in the formation of the brain–cerebrospinal fluid barrier

Elevated expression of glucose transporter‐1 in hypothalamic ependymal cells not involved in the formation of the brain–cerebrospinal fluid barrier

Glucose repression affects ion homeostasis in yeast through the regulation of the stress-activated ENA1 gene.

In this report we show that the ENA1/PMR2A gene is under glucose repression. The SNF1 protein kinase, acting independently from the HOG and calcineurin pathways, is essential to release ENA1 from glucose repression. The transcriptional repressor Ssn6p negatively regulates ENA1 expression and, like other glucose repressible genes, this repression is mediated in part by Mig1p. Deletion of a fragment from the ENA1 promoter that includes two Mig1p consensus binding sites gives a high level of expression in glucose without added salt. We suggest that regulation of ENA1 by the SNF1 pathway could be part of a general mechanism through which yeast cells respond to carbon source starvation by activating protective systems against different types of stress.

Genomic Footprinting of Mig1p in the MAL62 Promoter: BINDING IS DEPENDENT UPON CARBON SOURCE AND COMPETITIVE WITH THE Mal63p ACTIVATOR

Mig1p inhibits gene expression in glucose by binding the Cyc8p (Ssn6p)-Tup1p repressor to the promoter of glucose-repressible genes. While the binding properties of Mig1p have been studied in vitro and the ability of Mig1p-Cyc8p (Ssn6p)-Tup1p to repress has been studied in vivo, no experiments have measured the effect of a carbon source on the in vivo binding of Mig1p or the effect of bound MIg1p on activator occupancy of the upstream activation sequence (UAS). To obtain this information, we used genomic footprinting to investigate glucose repression of MAL62, a gene that is also regulated by the Mal63p activator. These experiments show that two interrelated mechanisms are involved in the glucose repression of MAL62: 1) competition between the Mal63p activator and Mig1p for DNA binding and 2) modulation of Mig1p binding by the carbon source. Mig1p affects basal MAL62 expression in the absence of Mal63p by binding to a site in the MAL62 promoter and affects Mal63p-dependent synthesis by also inhibiting the access of Mal63p to site 1 in the UASMAL. The binding of Mig1p is increased in glucose and decreased in nonrepressing sugars, but the increased binding in glucose is not due to an increase in the levels of Mig1p.

Heterologous Expression of the Serotonin 5-HT1A Receptor in Neural and Non-neural Cell Lines

Stable expression of neuronal receptors in cell lines of neural origin is important for studies of neurotransmitter mediated signal transduction. We have achieved this for the first time in three cell lines which are derived from various tissues of neural origin (hippocampus, HN2; chinese hamster brain explant, NCB-20; rat dorsal root ganglion, F-11). Following electroporation assisted transfer of a construct containing the hippocampal serotonin 5-HT1A receptor (5-HT1AR) DNA, one neural cell line, NG-108-15 (munne neuroblastoma x C6 glioma), failed to express the transfected activity, while three others as well as the non-neural CHO (chinese hamster ovary) cells expressed high levels of the receptor. Upon normalization to coexpressed human β-hexosaminidase B activity, it was found that the human 5-HT1AR, which is normally concentrated in the hippocampus and at a lesser density in the brain, was expressed at the highest level (15.7 × 104 receptors/cell) in the HN2 followed by the NCB-20 (8.3 × 104 receptors/cell), F-11 (4.4 × 104 receptors/cell) and lastly the non-neuronal CHO (4.2 × 104 receptors/cell) cells. Ten-twelve days after passage, a striking increase in expression of the receptor was observed only in the cell lines of neural origin. By contrast, there was no appreciable increase in expression of the transfected 5-HT1AR in the non-neural CHO cells over time. This late increase in expression was eliminated in cells which had been maintained in low glucose (1 g/L) for the first two days after passage, thus establishing a vital role of glucose in expression of the transfected 5-HT1AR in cell lines of neural origin. In all cases the 5-HT1AR was negatively coupled to adenylate cyclase, as evidenced by an agonist mediated decrease in prostaglandin E1 stimulated cyclic AMP levels.