Unraveling Insulin Transport Across the Brain Endothelium

Insulin acts on the brain and is transported across the blood-brain barrier (BBB). This route is the primary pathway for insulin’s entry into the brain as opposed to entry via CSF circulation. However, little is known regarding how circulating insulin crosses the BBB's highly-restrictive brain endothelial cell (BEC). Here, the mechanisms regulating BEC insulin uptake, signaling, and BBB transcytosis and how transport is affected by high-fat diet (HFD) feeding and astrocyte activity was examined. At physiologic insulin concentrations, blocking the insulin receptor (IR)—not the IGF-1 receptor—inhibited BEC insulin uptake and downstream insulin signaling. Inhibiting lipid raft endocytosis inhibited insulin uptake while blocking the insulin signaling to PI3-kinase or MEK had no effect. Isolated BECs (iBECs) from rats fed four wk of HFD had decreased insulin uptake and increased NFκB nuclear binding activity compared to iBECs from rats a normal chow diet (ND). Insulin-stimulated Akt and MEK phosphorylation, IR expression, and insulin degrading enzyme expression were comparable in HFD and ND iBECs. Using an in vitro BBB with co-cultured astrocytes and iBECs, insulin was found to be intact following BEC transcytosis and stimulating astrocytes with L-glutamate increased and L-NAME decreased insulin transcytosis. Thapsigargin treatment of astrocytes increased insulin transcytosis, suggesting that L-glutamate increases were mediated by increased cytosolic calcium in the astrocyte. In aggregate, this dissertation provides evidence for IR-specific, vesicle-mediated transport of intact insulin across the BEC. The effects of HFD feeding, nitric oxide inhibition, and astrocyte simulation suggest unique regulatory mechanisms govern BEC insulin uptake and transcytosis.