The Rho/Rac Guanine Nucleotide Exchange Factor Vav1 Regulates Hif-1α and Glut-1 Expression and Glucose Uptake in the Brain

Abstract

Vav1 is a Rho/Rac (Ras-related C3 botulinum toxin substrate) guanine nucleotide exchange factor expressed in hematopoietic and endothelial cells that are involved in a wide range of cellular functions. It is also stabilized under hypoxic conditions when it regulates the accumulation of the transcription factor HIF (Hypoxia Inducible Factor)-1α, which activates the transcription of target genes to orchestrate a cellular response to low oxygen. One of the genes induced by HIF-1α is GLUT (Glucose Transporter)-1, which is the major glucose transporter expressed in vessels that supply energy to the brain. Here, we identify a role for Vav1 in providing glucose to the brain. We found that Vav1 deficiency downregulates HIF-1α and GLUT-1 levels in endothelial cells, including blood-brain barrier cells. This downregulation of GLUT-1, in turn, reduced glucose uptake to endothelial cells both in vitro and in vivo, and reduced glucose levels in the brain. Furthermore, endothelial cell-specific Vav1 knock-out in mice, which caused glucose uptake deficiency, also led to a learning delay in fear conditioning experiments. Our results suggest that Vav1 promotes learning by activating HIF-1α and GLUT-1 and thereby distributing glucose to the brain. We further demonstrate the importance of glucose transport by endothelial cells in brain functioning and reveal a potential new axis for targeting GLUT-1 deficiency syndromes and other related brain diseases.

Keywords: GLUT-1; HIF-1α; Vav1; hypoxia; learning deficiency.

Figure 1

Vav1 regulates HIF-1α and GLUT-1 in endothelial cells. (A) HUVEC cells with control or Vav1 shRNA were incubated under normoxia or 1% O₂ conditions for 5 h, or (B) 16 h. Cells were lysed to measure HIF-1α, Vav1, and GLUT-1 levels by Western blotting. (C) hCMEC/d3 cells with control or Vav1 shRNA were incubated under normoxia or 1% O₂ conditions for 16 h. Cells were lysed to measure HIF-1α, Vav1, and GLUT-1 levels by Western blotting. (D) Control or Vav1 shRNA expressing hCMEC/d3 cells were transfected with an empty vector or HIF-1α overexpression vector. Cells were incubated under 1% O₂ conditions for 16 h and then subjected to Western blot. Each experiment was repeated three times, and representative data is selected. (* p < 0.05, ** p < 0.01, mean ± SD).

Figure 2

Vav1 regulates glucose uptake in endothelial cells. (A) HUVEC and hCMEC/d3 cells with control or Vav1 shRNA were starved in glucose-free medium for 1 h before the addition of 2-NBDG under hypoxic conditions with or without apigenin. After 1 h of incubation, cells were fixed with paraformaldehyde and observed by confocal microscopy. HUVEC (B) and hCMEC/d3 (C) cells with control or Vav1 shRNA in a 96-well plate were starved in glucose-free medium for 1 h and then treated with 2-NBDG with or without apigenin under hypoxic conditions for an hour. Mean fluorescence intensity (MFI) was measured to quantify 2-NBDG uptake levels (quadruplet mean ± SD). *** p < 0.001.

Figure 3

Vav1 deficiency delays glucose distribution in vivo. 8-week old female wild type or endothelial Vav1-deficient mice have fasted for 12 h in the presence of water. (A) Blood glucose levels were measured from the tail tip of each mouse (n = 10/group). (B) After 12 hours’ fasting, mice were given with 2 g/kg of glucose intraperitoneally. The blood glucose level was measured at each designated time point (n = 10/group). (C) The percentage of animals with 126 mg/dl or higher fasting blood glucose (FBG) in the basal state is displayed (n = 12/group). (D) The weight of mice measured after 12-h fasting (n = 12/group). Mean ± SD, * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 4

Vav1 deficiency downregulates glucose distribution in the brain. (A) The total lysate of mouse brain was subjected to Western blot to measure GLUT-1 protein expression levels (n = 3/group). Mean ± SD, *** p < 0.001. (B) Wild type or endothelial Vav1-deficient mice were subjected to a PET/CT scan to measure the distribution of 18F-FDG. Mice fasted for 12 h before intravenous injection of 18F-FDG (Arrows indicate brain hemispheres). One hour after 18F-FDG injection, the whole body of mice was scanned. The scanned signal in brains was normalized and then numerated as mean (C) and total (D) standardized uptake value body weight (SUVbw) (n = 10/group). (E) Wild type or endothelial Vav1-deficient mice were subjected to a fear conditioning test. Both contextual and cued conditions were given to measure the percentage of time in a frozen state (n = 10/group). Mean ± SD, * p < 0.05. All mice were female and eight weeks old.