Immortalized Mouse Cerebral Capillary Endothelial Cells (cEND)

Notch Signaling Regulates Glut1 Expression and Glucose Metabolism in BMECs In Vitro

High-fat diet (HFD)-induced obesity impairs brain glucose uptake and blood-brain barrier (BBB) function. Glut1, a key transporter of brain glucose uptake, is downregulated in brain endothelial cells from obese mice. However, the mechanism for this dysregulation remains poorly understood. In the present study, Zhu et al. examined the hypothesis that Notch signaling mediates HFD-induced changes in BBB permeability, glucose uptake, and insulin signaling.

In situ RNA analysis and ribosomal profiling revealed that short-term HFD feeding downregulated Glut1 and Notch signaling in brain microvascular endothelial cells (BMECs). They next tested if downregulation of Notch signaling was a compensatory response to the reduced glucose transport and glycolysis in BMECs isolated from HFD-fed mice. Immortalized cerebral endothelial (cEND) cells were treated with a Glut1 inhibitor (WZB117) or vehicle and then analyzed by mRNA sequencing. Inhibition of Glut1 did not alter Notch signaling, suggesting that downregulation of Notch signaling is not a secondary response to reduced glucose uptake. They then tested the effect of Notch signaling on Glut1 regulation in cEND cells. Activation of Notch signaling with Dll4 increased glycolysis (Fig. 1A and B). Incubation of cEND cells with serum from HFD-fed mice for 24 h decreased Glut1 protein expression relative to cells incubated in serum from control diet (CD)-fed mice (Fig. 2A and 2B). However, co-incubation with Dll4 rescued Glut1 expression (Fig. 2A and 2B). Similarly, glycolytic capacity and glycolytic reserve were decreased in cEND cells incubated with serum from HFD-fed mice and restored by Notch activation (Fig. 2C and 2D). Together, these results demonstrate that Dll4-mediated Notch activation can rescue the HFD serum-induced inhibition of Glut1 expression and glycolysis in vitro.

Fig. 1. Notch-activation promotes glycolytic flux in cEND cells (Zhu Y Y, Mehlkop O, et al., 2025).

Fig. 2. Notch signaling regulates Glut1 expression in vitro (Zhu Y Y, Mehlkop O, et al., 2025).

Concentration-Dependent Induction of Cld-5 Expression by E2 and DPN

Breast cancer brain metastasis (BCBM) is a devastating and deadly disease that has few treatment options. The blood–brain barrier (BBB) is an effective barrier to circulating metastatic cancer cells and estrogen receptor β (ERβ) has been shown to upregulate tight junction proteins. To gain further insight, Kirchner et al.examined 17β-estradiol (E2) and the selective ERβ agonist diarylpropionitrile (DPN) in endothelial and cancer cells, using Western blot analysis and in vitro BBB models.

To evaluate the impact on claudin-5 (Cld-5), an important estrogen target that is critical to BBB integrity, the authors performed Western Blot on murine (cEND) and human (hCMEC/D3) brain endothelial cells. After 24 h, E2 (10⁻¹⁰ M) increased Cld-5 expression by 1.20 ± 0.12-fold in cEND cells (p = 0.10) (Fig. 3a). Other E2 concentrations (10⁻¹² M, p = 0.89; 10⁻⁸ M, p = 0.28; 10⁻⁶ M, p = 0.50) and DPN concentrations (10⁻¹² M, p = 0.76; 10⁻¹⁰ M, p = 0.87; 10⁻⁸ M, p = 0.66; 10⁻⁶ M, p = 0.82) did not significantly alter Cld-5 expression (Fig. 3a and b). In hCMEC/D3 cells, E2 and DPN treatments showed no significant differences (Fig. 3c and d). However, 10⁻¹² M DPN significantly decreased Cld-5 expression by 0.79 ± 0.11-fold (p = 0.50) (Fig. 3d).

Fig. 3. Western blot analysis showing the concentration-dependent induction of claudin-5 (Cld-5) expression by E2 and DPN. The endothelial cell lines cEND and hCMEC/D3 were analyzed for 24 h in the concentrations 10⁻¹², 10⁻¹⁰, 10⁻⁸, and 10⁻⁶ M with E2 or DPN (Kirchner J, Völker E, et al., 2024).