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The Natural Protective Mechanism Against Hyperglycemia in Vascular Endothelial Cells

OBJECTIVE Vascular endothelial cells (VECs) downregulate their rate of glucose uptake in response to hyperglycemia by decreasing the expression of their typical glucose transporter GLUT-1. Hitherto, we discovered critical roles for the protein calreticulin and the arachidonic acid–metabolizing enzyme 12-lipoxygenase in this autoregulatory process. The hypothesis that 4-hydroxydodeca-(2E,6Z)-dienal (4-HDDE), the peroxidation product of 12-lipoxygenase, mediates this downregulatory mechanism by activating peroxisome proliferator–activated receptor (PPAR) δ was investigated. RESEARCH DESIGN AND METHODS Effects of 4-HDDE and PPARδ on the glucose transport system and calreticulin expression in primary bovine aortic endothelial cells were evaluated by pharmacological and molecular interventions. RESULTS Using GW501516 (PPARδ agonist) and GSK0660 (PPARδ antagonist), we discovered that high-glucose–induced downregulation of the glucose transport system in VECs is mediated by PPARδ. A PPAR-sensitive luciferase reporter assay in VECs revealed that high glucose markedly increased luciferase activity, while GSK0660 abolished it. High-performance liquid chromatography analysis showed that high-glucose incubation substantially elevated the generation of 4-HDDE in VECs. Treatment of VECs, exposed to normal glucose, with 4-HDDE mimicked high glucose and downregulated the glucose transport system and increased calreticulin expression. Like high glucose, 4-HDDE significantly activated PPARδ in cells overexpressing human PPAR (hPPAR)δ but not hPPARα, -γ1, or -γ2. Moreover, silencing of PPARδ prevented high-glucose–dependent alterations in GLUT-1 and calreticulin expression. Finally, specific binding of PPARδ to a PPAR response element in the promoter region of the calreticulin gene was identified by utilizing a specific chromatin immunoprecipitation assay. CONCLUSIONS Collectively, our data show that 4-HDDE plays a central role in the downregulation of glucose uptake in VECs by activating PPARδ.

calreticulin gene (F, forward; R, reverse; numbers indicate base-pair positions relative to the beginning of the 3,000 bp 5'-untranslated region) were:

SUPPLEMENTAL FIGURES
Supplemental Figure S4. High glucose and GW501516 selectively reduce GLUT-1, but not GLUT-3 and GLUT-4, content in VEC. VEC were incubated with 5.5 or 25 mmol/L glucose for 48 h, without or with 100 nmol/L of GW501516. The cells were then washed, lysed and taken for Western blot analyses of the various GLUTs and α-tubulin. The latter served as a protein loading control.
Supplemental Figure S5. PPARδ δ δ δ antagonistic activity of GSK0660 in a 3xPPRE-TK-Luciferase reporter assay. VEC cultures at 2 mmol/L glucose were transfected with the 3xPPRE-TK-Luciferase reporter plasmid and the Renilla luciferase plasmid and then incubated for 24 h with 1 nmol/L of GW501516 and/or 1 µmol/L of GSK0600, as indicated. The cells were then washed, harvested, lysed and taken for the luciferase activity assay. Results are given as the relative increase of luciferase activity in comparison with the 2 mmol/L glucose (vehicle) incubation. P <0.05 for differences from the 2 mmol/L glucose incubation *without or ** with GW501516 (n=4). Figure S6. Overexpression of hPPARα, -γ1, -γ2 and -δ δ δ δ in VEC. VEC cultures were transfected with the following expression plasmids: pSG5-hPPARα or pCMX-hPPARγ1 or pCMX-hPPARγ2 or pcDNA-hPPARδ. Control cells were transfected with the corresponding empty plasmids. Cells were also co-transfected with pSVPORT-hRXR, pEGFP-N1 plasmid, 3xPPRE-TK-Luciferase reporter plasmid and the Renilla luciferase plasmid. After 48 h the cells were harvested, lysed and taken for a Western blot analyses with specific antibodies against the various hPPAR isotypes. Figure S7. Effects of 4-HDDE on the viability of VEC. Confluent VEC cultures that had been pre-conditioned at 5.5 mmol/L glucose received the same fresh medium without or with the indicated concentrations of 4-HDDE and were incubated for 48 h. The cultures were then washed 3 times with PBS at room temperature, lysed and taken for protein determination. The protein content in the absence of 4-HDDE (0.21 ± 0.03 mg/well) was taken as 100% (n=4).

Supplemental Figure S8. HPLC profiles of 4-HDDE analysis.
Representative HPLC tracings of 4-HDDE in extracts of cell culture media (A) and rat plasma samples (B). Arrows indicate peaks of 4-HDDE, as resolved with the pure compound.
Supplemental Figure S9. The autoregulation of glucose transport system in EA.hy926 cells. Confluent EA.hy926 cultures were exposed to 5.5 or 25 mmol/L glucose for 48 h. (A, B), in the absence or presence of 1 µmol/L of GSK0660, then washed and taken for Western blot analysis of GLUT-1 and Calreticulin. (C, D) Similar cells were incubated with 1 µmol/L of GSK0660 and/or 4-HDDE, then washed and taken for the standard [ 3 H]dGlc uptake assay. The rate of dGlc