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Endothelial dysfunction leading to cardiovascular disease risk involves a decrease in nitric oxide (NO) production. In physiological conditions shear stress is a potent stimulation of endothelium-derived NO production and flow mediated NO production is regulated by the activation of endothelial NO synthase (eNOS). In endothelial cells, eNOS, aldose reductase (AR), a rate limiting enzyme of polyol pathway, and glutathione reductase (GR) share a NADPH as an obligate cofactor. In diabetec condition intracellular polyol pathway is activated and this may decrease shear stress-induced endothelial NO production and increase intracellular oxidative stress via inhibition of eNOS and GR by NADPH consumption. Therefore we investigated whethter AR inhibitor epalrestat improved endothelial NO production under high glucose condition to elucidate the mechanism of endothelial dysfunction in diabetes. We incubated human umbilical vein endothelial cells (HUVECs) in normal (5mM) and high (30mM) glucose condition for 72 hours, with or without epralrestat, or 100U/ml superoxide dismutase (SOD), respectively. After exchange of medium for Krebs’ buffer, HUVECs were exposed to 12dyne/cm2 steady laminar fluid shear stress for 5 minutes. NO release from HUVECs was measured as NO2 using a NOx analyzing HPLC system by Griess reaction. Next we harvested the cells in lysis buffer and analyzed phosphorylation of Akt (shear induced intracellular signal transduction) and eNOS by western blotting, and measured intracellular 8-OHdG and ratio of NADPH/NADP. In high glucose condition NO2 was decreased and 8-OHdG increased compared to low glucose. NO2 was restored and 8-OHdG was reduced by epalrestat significantly (p These results showed that polyol pathway activation plays a key role in endothelial NO production under high glucose condition via a cofactor NADPH.

Background: Adipose tissue inflammation induces inflammatory molecules production leading to insulin resistance and increased cardiovascular disease risk. Recently it has become apparent that innate immune system is activated by saturated fatty acids (SFAs) by a process involved in TLR4 signaling. HDL and apoA-I has anti-inflammatory effects, however the mechanisms are not well understood in adipocytes. Aims: We examined to elucidate the effect of HDL and/or ApoA-I mediated cholesterol efflux on induction of inflammatory gene expression by palmitate in adipocytes via TLR4 dependent immune system. Methods: 3T3-L1 adipocytes are exposure to 250uM palmitate for periods of up to 24 hours with or without HDL or Apo A-I. Antisense directed to ABCG-1 or ABCA-1 is added 24 hours prior to each reagent challenge. Cells were harvested and estimated for expression of Saa 3 gene using real-time PCR or detection of TLR4 in raft or non raft by western blotting. Lipid rafts were purified by ultracentrifugation on discontinuous sucrose gradient, and identified by the presence of caveolin-1. Results: TLR4 was translocated into lipid raft from non raft within 15 minutes and dislocated by 60 minutes after palmitate exposure. Saa 3 gene expression was significantly up-regulated by 24 hours palmitate exposure in adipocytes. TLR4 translocation to raft and Saa 3 gene up- regulation induced by palmitate was significantly inhibited by pretreatment of HDL or Apo A-I for 6 hours. Antisense directed to ABCG-1 or SRB1 abrogated these inhibitory effects by HDL, but not ApoA-I whereas antisense to ABCA-1 did by Apo A-I but not HDL. Conclusions: These findings suggest that TLR4 mediates palmitate-induced Saa 3 gene expression in adipocytes. A reduction in membrane cholesterol mediated by ABCG-1, SRB1 or ABCA-1 can account for the ability of HDL or ApoA-I to disrupt stimulation of inflammatory cytokines by palmitate