Hyperinsulinemia alters Notch1 and ZO-1 expression in human brain microvascular endothelial cells

Abstract

Alzheimer’s disease (AD) is an irreversible disease of aging that progressively impairs cognitive function. Although the pathogenesis of AD remains elusive, dysfunction of the blood-brain barrier (BBB) appears to be an important contributing factor. Of the cells that form the BBB, brain microvascular endothelial cells play an important role in maintaining the integrity of the barrier. Disruption of endothelial tight junctions may contribute to the BBB dysfunction observed in AD. Complete understanding of how this may occur remains unclear, but the intersection between AD and type 2 diabetes mellitus (T2DM) suggests possibilities. Individuals with T2DM are at greater risk of developing AD, and the hyperinsulinemia that characterizes untreated T2DM may contribute to endothelial damage and BBB dysfunction by disrupting tight junctions. The cellular mechanisms by which this dynamic occurs continue to be explored, but one signaling pathway that may be involved is the Notch signaling pathway. The objective of this study was to investigate the effects of hyperinsulinemia on Notch1 and zonula occludens-1 (ZO-1, a key tight junctional protein) expression in human brain microvascular endothelial cells. We hypothesized hyperinsulinemia disrupts tight junctions in brain endothelial cells by decreasing ZO-1 protein expression in a Notch1-dependent manner. To explore this hypothesis, human cerebral microvascular endothelial cells (hCMEC/d3 cell line) were exposed to hyperinsulinemic conditions (100 nM) for 0.5, 1, 2, and 4 hours. Following treatment, total cellular protein was isolated, quantified, and separated by SDS-PAGE. Notch1 and ZO-1 protein expression were analyzed by western blot, and expression levels normalized to GAPDH. Our results indicate hyperinsulinemia increased protein expression of Notch1 by almost 5-fold and decreased ZO-1 expression by 3-fold in a time-dependent manner compared to untreated control cells. These results demonstrate hyperinsulinemia alters both Notch1 and ZO-1 protein expression along a parallel timeline, suggesting ZO-1 expression may be dependent on Notch1 signaling; however, further studies are needed to determine whether Notch1 signaling is required for decreased ZO-1 expression during hyperinsulinemic conditions. Disruption of cerebral microvascular endothelial tight junctional integrity due to hyperinsulinemia in T2DM may negatively impact BBB functionality, lending insight into the development and progression of AD. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.