Activation of the unfolded protein response in high glucose treated endothelial cells is mediated by methylglyoxal

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/10 6 cells, P < 0.01) and formation of MG-modified proteins (24.0 ± 3.7 versus 14.1 ± 3.2 pmol/10 6 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.

Hexokinase-2 Glycolytic Overload in Diabetes and Ischemia–Reperfusion Injury

“…Selective increase in HK2 was discovered by quantitative characterization of the cytosolic proteome by label-free quantitative proteomics analysis and validated by western blotting. This was associated with increased flux of glucose metabolism [14], corroborating outcomes from previous metabolic tracer studies [21]. A metabolic marker of metabolic dysfunction was increased flux of formation of methylglyoxal (MG) and MG-derived advanced glycation end-products (AGEs).…”

“…VDAC provides a conduit for HK2 to access ATP from the intramembrane space of mitochondria for G6P formation [105]. HK2 displacement by increased concentrations of G6P [14,106], twofold increase in HAECs in hyperglycemia [107], and three-to tenfold increase in myocardial tissue in ischemia [32,33], impairs ATP utilization, and impairs oxygen consumption analogous to that found under conditions of inhibition of ADP recycling. HK2 is only weakly product inhibited by G6P and its activity in situ is maintained when G6P concentration is abnormally in high in glycolytic overload; so increased formation of G6P continues, exacerbating and prolonging mitochondrial dysfunction [2].…”

“…Recent research suggests that abnormal increase of HK2 plays a key role when control of glycolysis is impaired, as in unscheduled glycolysis. In this case, abnormal increase in G6P and downstream glycolytic intermediates are key players in the tissue-specific chronic pathogenesis of hyperglycemia associated with diabetes, ischemia-reperfusion injury, and potentially, in cell senescence [14][15][16]. We call this damaging condition glycolytic overload.…”

“…[101,102]. Stabilization of HK2 to degradation was found in response to high cytosolic glucose concentration in HAECs in primary culture [14]. HK2 has a turnover fivefold higher than that of HK1, so change in HK2 protein abundance may have a marked effect on total hexokinase activity; both hexokinases often operate in situ under glucose substrate saturation conditions [2].…”

“…(iv) Increased DHAP and GA3P leading to increased formation and concentration of MG, with related formation of MGderived AGEsthe dicarbonyl stress pathway [45]. Increased MG-derived AGEs produce increased protein misfolding and thereby activate the UPR in the cytosolic and endoplasmic reticulum, including increasing downstream inflammatory and prothrombotic mediators [14]. (v) Increased glycogen deposition -HK2 displacement from mitochondria increases glycogen synthesis through metabolic channeling of glucose to glycogen synthesis [106].…”