Abstracts

Rationale: Glut-1 facilitates glucose transport across the blood brain barrier. This protein is over-expressed on brain micovascular endothelial cells and astrocyte membranes. Glut-1 deficiency syndrome is an autosomal-dominant disorder characterized by infantile seizures, developmental delay, acquired microcephaly, ataxia and spasticity and is caused by GLUT 1 haploinsuffiency. Upregulation of the wild-type GLUT-1 allele could mitigate the haploinsufficient state. Valproic acid (VPA), one of the widely used antiepileptic drugs, was recently found to inhibit histone deacetylase (HDAC). HDAC inhibitors increase acetylation of histones and other proteins, relaxing the tertiary structure of chromatin and facilitating access of the transcriptional machinery to target genes. Manipulation of histone acetylation has been shown to alter transcription in approximately 2% of genes. By increasing histone acetylation at the GLUT-1 promotor, VPA could increase GLUT-1 gene expression. This study was designed to evaluate the effects of VPA on glucose transport in astrocyte cultures derived from GLUT-1 heterozygous (HET) mice. Methods: GLUT-1+/-(HET) mice were mated with wild-type (WT) mice. One day (P1) old mice were decapitated and tails were removed for cell culture and genotyping. Primary astrocyte cultures were prepared from cerebral cortex of P1 neonatal mice through a modification of the method designed by Vega et al. (2002). After 7 days of primary culture, 5x10⁵cells/well were seeded in 12 well plates and grown for 48 h (confluent) with 25 mM glucose medium. Cultured astrocytes were incubated with VPA (0.05, 0.5, 5 mM) for 48 h. On day 3, glucose uptake capacity of astrocytes was measured by using C14-2-DOG under zero-trans conditions. Results: Glucose uptake was significantly lower in Glut 1 HET astrocytes compared to WT astrocytes at 1, 5, 10, 30 and 60 minutes. Subsequent studies used the 1 minute time point as the differences were most significant (HET 53.73% of WT). The HET astrocyte Vmax for glucose transport was significantly lower and the Km was the same compared to the WT astrocytes, consistent with a gene dosage effect.The HET astrocyte glucose uptake treated with VPA (0.05 and 0.5 mM) for 48 hours was significantly increased compared to untreated control HET astrocytes. HET untreated control was 3.03±0.14, 0.05mM VPA was 4.19±0.17, and 0.5mM VPA was 4.27±0.15 pmol/min/ug. These results were confirmed by western blot. GLUT-1 expression was increased in the treated HET astrocytes compared to the untreated HET astrocytes. Conclusions: Our findings demonstrate that VPA, a known HDAC inhibitor, encreases glucose transport capacity in GLUT-1 HET cerebal astrocytes. The GLUT-1 stimulating properties of VPA make this drug a treatment candidate for epileptic patients with the GLUT-1 deficiency syndrome