IDF21-0162 Hexokinase-2 linked glycolytic overload – key initiating driver of the development of diabetic vascular complications

Abstract

Listen

Translate article

Background: The hypothesis of multiple pathways of metabolic dysfunction in hyperglycemia of diabetes explains involvement of mitochondrial dysfunction, protein kinase c, hexosamine pathway and methylglyoxal (MG)-derived formation of advanced glycation endproducts in the cell biology of vascular complications of diabetes. It does not explain, however, how the increased glucose metabolism driving this dysfunction occurs at sites of vascular complications when the hexokinase isozymes therein, hexokinase-1 (HK1) and hexokinase-2 (HK2), are saturated with glucose in normoglycemia and there is no increase of hexokinase expression in hyperglycemia. This conundrum was recently resolved when it was found that high cellular glucose concentration stabilizes HK2 to proteolysis, increasing HK2 protein and activity. This sends a wave of increased glycolytic intermediates through early-stage glycolysis driving metabolic dysfunction. Increased HK2 may be corrected by glyoxalase 1 inducer, trans-resveratrol and hesperetin in combination (tRES-HESP). HK2-linked glycolytic overload is the initiator of metabolic dysfunction in hyperglycemia and tRES-HESP may offer an exciting new route to treatment of diabetic complications. Aim: The aim of this study is to assess the evidence for HK2-linked glycolytic overload in human aortal endothelial cells (HAECs) and renal proximal tubular epithelial cells (PTECs) in model hyperglycemia in vitro and explore literature evidence for HK2-linked glycolytic overload sites of vascular complications in vivo. Method: HAECs and PTECs were cultured according to the supplier’s instructions in 4.1 - 20 mM glucose and 7 – 25 mM glucose respectively for 6 - 72 h. HK2 protein abundance was assessed by Western blotting, normalized to β-actin; and glucose consumption and flux of formation of MG (assessed by accumulation of D-lactate) were determined by assay of glucose and D-lactate at the start and end of cultures, normalizing flux to cell number. Results: HK2 protein increased progressively with time and increasing glucose concentration in HAEC and PTEC cultures. In HAECs, HK2 abundance was increased maximally by 37% whereas total glucose consumption was increased by 59% (6.77 ± 0.12 to 10.75 ± 0.21 µmol/day/million cells; P<0.001, n = 3). HK2 abundance increased from 4.1 mM to 20 mM glucose, increasing ca. 2.4% per mM glucose. HK2 abundance correlated positively with initial glucose concentration and flux of glucose consumption (r = 0.96 and r = 0.85, respectively; P<0.001; n = 12, Pearson) and metabolic dysfunction – as judged by increased flux of MG formation (r = 0.93; P<0.001, n = 12); HK1 abundance did not. tRES-HESP prevented increased HK2 abundance, increased glucose consumption and metabolic dysfunction of HAECs in high glucose concentration. Discussion: We conclude that HK2 abundance increases with increased glucose concentration in the clinical range in diabetes in HAECs and PTECs, producing glycolytic overload and metabolic dysfunction in high glucose concentration. Criteria for HK2-linked glycolytic overload – GLUT1 and/or GLUT3 glucose uptake and expression of HK2 - are found at sites of diabetic vascular complications clinically; and a biomarker of its occurrence - increased glycogen deposition - is found with development of complications. tRES-HESP offers a prospective new approach to prevent the development of vascular complication of diabetes.