Effects of Diabetes‐Like Altered Glycemia on BBB Gating Functions: Impact on Efflux Transporters (LB600)

Abstract

Altered glycemic conditions, such as those observed in diabetic patients, impair blood‐brain barrier (BBB) endothelial physiology and serve as potential risk factor for the onset and progression of major CNS disorders (such as Alzheimer’s disease). In this study, we assessed the impact of diabetes‐like hyper and hypoglycemic conditions on the expression and functionality of major BBB efflux transporters (P‐glycoprotein and MRP4). Confluent monocultures of hCMEC/D3 endothelial cells (a well characterized human BBB endothelial cell line) were exposed to normal (5.5mM), hypo (2.2mM) ‐ or hyper (35mM)‐glycemic conditions of D‐glucose for 3, 6, 12 and 24 h. Following treatment, cells were either lysed or fixed for subsequent protein expression and distribution analysis by western blotting and immunofluorescence. Further, parallel hCMEC/D3 cultures were chronically exposed to identical experimental conditions, but for a period of 3 days, and profiled for genome‐wide transcriptional changes using Illumina® DNA microarray. Our results indicate an up‐regulation of P‐glycoprotein (P‐gp) and MRP4 protein following 12 ‐24 h acute exposure to hypoglycemia (~1.1 fold increase, vs. control), while no differences were observed at 3 h. Hyperglycemic exposure resulted in a time‐dependent alteration of P‐glycoprotein and MRP4 expression with an initial increase at 3 h followed by restoration to normal (MRP4) or down‐regulation (P‐glycoprotein). These results were further confirmed by using L‐glucose as an osmotic control. Gene chip analysis revealed a moderate increase of P‐gp and MRP4 gene expression following repeated exposures to hyperglycemia. In contrast, chronic exposure to cycles of hyper ‐ hypoglycemia significantly decreased P‐gp gene expression (≍ 50% vs. control) while the MRP4 expression was mostly unaffected. In summary, our data demonstrate that acute or recurrent exposures to hypo‐ and hyperglycemia potentially alter the protein/gene expression of major BBB efflux transporters that would impact the BBB integrity and gating function as well as brain drug disposition. Additional studies are currently in progress to determine the changes in the functionality of these transporters in relation to their altered expression.Grant Funding Source: Supported by A.R.D.F and in part by NIH/NIDA R01‐DA029121‐01A1 to LC