485-P: Combination of Trans-resveratrol and Hesperetin Corrects Unscheduled Increased Glucose Metabolism Driving Endothelial Cell Dysfunction in Hyperglycemia In Vitro

Endothelial dysfunction contributes to the development of vascular complications of diabetes. Hyperglycemia induces multiple pathways of metabolic dysfunction: mitochondrial dysfunction, hexosamine and protein kinase C pathway activation and increased formation of methylglyoxal (MG). We recently showed that increased glucose metabolism driving this is produced by glucose-induced stabilization of hexokinase-2 (HK2) to proteolysis, increasing HK2 activity. In this study we investigated correction of this by combination of resveratrol and hesperetin (tRES-HESP) which induces expression of glyoxalase 1 (Glo1) and protects against increased MG. Human aortal endothelial cells (HAECs) were incubated in primary culture with 5 mM or 20 mM glucose for 6 days with and without 10 µM tRES-HESP. Glucose consumption and flux of MG formation was measured by enzymatic assay. Activity of glyoxalase 1 (Glo1) was measured by spectrophotometric assay. HK2 protein was determined by Western blotting. The flux of glucose consumption in high glucose concentration cultures of HAECs was increased, compared to low glucose concentration control (1647 ± 242 vs. 748 ± 150 nmol/10⁶ cells/day, n = 8; +120%, P<0.01). Flux of formation of MG was increased similarly: 1.13 ± 0.10 v 0.62 ± 0.07 nmol/10⁶ cells/day, n = 8; +81%, P<0.01). Glo1 activity was decreased in high glucose culture: 1822 ± 143 vs. 1370 ± 160 mU/mg protein (-25%, P<0.01). HK2 protein was increased: +94% at day 3 and + 58% at day 6 (P<0.001). tRES-HESP corrected increased glucose consumption and MG formation, and decreased Glo1 activity in high glucose concentration cultures. This was achieved by inducing expression of glucose-6-phosphate dehydrogenase (G6PD), decreasing HK2 expression by decreasing the glucose-6-phosphate (G6P) and related carbohydrate response element transactivation by G6P/Mondo A/Mlx. tRES-HESP offers a new effective therapy of endothelial dysfunction.