Abstract
Metabolic dysfunction of endothelial cells in hyperglycemia contributes to the development of vascular complications of diabetes where increased reactive glycating agent, methylglyoxal (MG), is involved. We assessed if increased MG glycation induced proteotoxic stress, identifying related metabolic drivers and protein targets. Human aortal endothelial cells (HAECs) were incubated in high glucose concentration (20 mM versus 5 mM control) in vitro for 3–6 days. Flux of glucose metabolism, MG formation and glycation and changes in cytosolic protein abundances, MG modification and proteotoxic responses were assessed. Similar studies were performed with human microvascular endothelial HMEC-1 cells where similar outcomes were observed. HAECs exposed to high glucose concentration showed increased cellular concentration of MG (2.27 ± 0.21 versus 1.28 ± 0.03 pmol/106 cells, P < 0.01) and formation of MG-modified proteins (24.0 ± 3.7 versus 14.1 ± 3.2 pmol/106 cells/day; P < 0.001). In proteomics analysis, high glucose concentration increased proteins of the heat shock response – indicating activation of the unfolded protein response (UPR) with downstream inflammatory and pro-thrombotic responses. Proteins susceptible to MG modification were enriched in protein folding, protein synthesis, serine/threonine kinase signalling, glycolysis and gluconeogenesis. MG was increased in high glucose by increased flux of MG formation linked to increased glucose metabolism mediated by proteolytic stabilisation and increase of hexokinase-2 (HK-2); later potentiated by proteolytic down regulation of glyoxalase 1 (Glo1) - the major enzyme of MG metabolism. Silencing of Glo1, selectively increasing MG, activated the UPR similarly. Silencing of HK-2 prevented increased glucose metabolism and MG formation. trans-Resveratrol and hesperetin combination (tRES-HESP) corrected increased MG and glucose metabolism by increasing expression of Glo1 and decreasing expression of HK-2. Increased MG glycation activates the UPR in endothelial cells and thereby may contribute to endothelial cell dysfunction in diabetic vascular disease where tRES-HESP may provide effective therapy.
Highlights
Increased plasma glucose concentration in diabetes induces dysfunction of endothelial cells (ECs) linked to development of diabetic vascular complications – nephropathy, retinopathy, peripheral neuropathy, generalised microangiopathy and increased risk of cardiovascular disease
We studied the effect of knockdown of HK-2 on glucose metabolism and MG formation in Human aortal endothelial cells (HAECs) in high glucose concentration
We considered if dicarbonyl stress and increased glucose consumption of HAECs in high glucose could be reversed by small molecule therapeutic agents
Summary
Increased plasma glucose concentration in diabetes induces dysfunction of endothelial cells (ECs) linked to development of diabetic vascular complications – nephropathy, retinopathy, peripheral neuropathy, generalised microangiopathy and increased risk of cardiovascular disease This is characterized by increased inflammatory signalling, expression of adhesion molecules and secretion of inflammatory cytokines, apoptosis and processes supporting atherosclerosis[1,2,3,4,5]. When incubated in high glucose concentration, they have: increased cellular MG concentration and increased MG-H1 content of cell protein; increased production of inflammatory mediators, extracellular matrix proteins and adhesion molecules and apoptosis; and impaired angiogenesis Many of these effects were prevented by overexpression of Glo[1] and exacerbated by siRNA silencing and chemical inhibition of Glo[14,5,12,13]. The aim of this study is to test this hypothesis and identify the metabolic drivers for increased MG concentration in ECs in high glucose concentration, proteins susceptible to glycation by MG and the pathways in which high glucose-induced changes of protein abundance and MG-modified proteins are enriched
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