Unravelling the regulation of insulin transport across the brain endothelial cell

Abstract

Aims/hypothesis: For circulating insulin to act on the brain it must cross the blood–brain barrier (BBB). Remarkably little is known about how circulating insulin crosses the BBB’s highly restrictive brain endothelial cells (BECs). Therefore, we examined potential mechanisms regulating BEC insulin uptake, signalling and degradation during BEC transcytosis, and how transport is affected by a high-fat diet (HFD) and by astrocyte activity. Methods: 125I–TyrA14-insulin uptake and transcytosis, and the effects of insulin receptor (IR) blockade, inhibition of insulin signalling, astrocyte stimulation and an HFD were tested using purified isolated BECs (iBECs) in monoculture and co-cultured with astrocytes. Results: At physiological insulin concentrations, the IR, not the IGF-1 receptor, facilitated BEC insulin uptake, which required lipid raft-mediated endocytosis, but did not require insulin action on phosphoinositide-3-kinase (PI3K) or mitogen-activated protein kinase kinase (MEK). Feeding rats an HFD for 4 weeks decreased iBEC insulin uptake and increased NF-κB binding activity without affecting insulin PI3K signalling, IR expression or content, or insulin degrading enzyme expression. Using an in vitro BBB (co-culture of iBECs and astrocytes), we found insulin was not degraded during transcytosis, and that stimulating astrocytes with l-glutamate increased transcytosis, while inhibiting nitric oxide synthase decreased insulin transcytosis. Conclusions/interpretation: Insulin crosses the BBB intact via an IR-specific, vesicle-mediated transport process in the BECs. HFD feeding, nitric oxide inhibition and astrocyte stimulation can regulate BEC insulin uptake and transcytosis.