High glucose-induced oxidative stress increases transient receptor potential channel expression in human monocytes

Abstract

Objective: Transient receptor potential (TRP) channel-induced cation influx activates human monocytes, which play an important role in the pathogenesis of atherosclerosis. In the present study, we investigated the effects of high glucose-induced oxidative stress on TRP channel expression in human monocytes.

Research design and methods: Human monocytes were exposed to control conditions (5.6 mmol/l d-glucose), high glucose (30 mmol/l d-glucose or l-glucose), 100 micromol/l peroxynitrite, or high glucose in the presence of the superoxide dismutase mimetic tempol (100 micromol/l). TRP mRNA and TRP protein expression was measured using quantitative real-time RT-PCR and quantitative in-cell Western assay, respectively. Calcium influx and intracellular reactive oxygen species were measured using fluorescent dyes.

Results: Administration of high d-glucose significantly increased reactive oxygen species. High d-glucose or peroxynitrite significantly increased the expression of TRP canonical type 1 (TRPC1), TRPC3, TRPC5, TRPC6, TRP melastatin type 6 (TRPM6), and TRPM7 mRNA and TRPC3 and TRPC6 proteins. High d-glucose plus tempol or high l-glucose did not affect TRP expression. Increased oxidative stress by lipopolysaccharide or tumor necrosis factor-alpha increased TRP mRNA expression, whereas the reduction of superoxide radicals using diphenylene iodonium significantly reduced TRP mRNA expression. Increased TRPC3 and TRPC6 protein expression was accompanied by increased 1-oleoyl-2-acetyl-sn-glycerol-induced calcium influx, which was blocked by the TRPC inhibitor 2-aminoethoxydiphenylborane. TRPC6 mRNA was significantly higher in monocytes from 18 patients with type 2 diabetes compared with 28 control subjects (P < 0.05).

Conclusions: High d-glucose-induced oxidative stress increases TRP expression and calcium influx in human monocytes, pointing to a novel pathway for increased activation of monocytes and hence atherosclerosis in patients with diabetes.

FIG. 1.

Changes of mRNA expression of (A) TRPC3, (B) TRPC6, (C and D) TRPC1, TRPC5, TRPM6, TRPM7, and TNF-α induced by high glucose and ONOO. Exposure of monocytes to high glucose (HG; 30 mmol/l d-glucose) or ONOO (100 μmol/l) for 4 h significantly increased the TRPC3 and TRPC6 mRNA expression. The effect of high glucose is attenuated by superoxide dismutase mimetic TMP (100 μmol/l). Administration of l-glucose showed no significant effect. Control (open bar) indicates control glucose (5.6 mmol/l d-glucose). The expression of each factor is normalized to GAPDH expression. Data are mean ± SEM from at least four to six independent experiments. *P < 0.05; **P < 0.01 compared with control conditions. E: Bar graph showing TRPC3 mRNA and TRPC6 mRNA expression (normalized ratio) in monocytes from 28 age-matched control subjects (open bars) and from 18 patients with type 2 diabetes (filled bars). *P < 0.05 compared with control.

FIG. 2.

Quantitative in-cell Western assay of TRPC3 and TRPC6 channel protein expression in monocytes treated with HG (30 mmol/l d-glucose) in the absence and presence of TMP (100 μmol/l), ONOO (100 μmol/l), l-glucose (30 mmol/l), or control (5.6 mmol/l d-glucose). A: Representative conventional Western assay (TRPC3 protein and GAPDH protein for loading control with top values indicating densitometric analysis of blots). The TRPC3/GAPDH ratio (fold over control) is also indicated. B and D: Representative in-cell Western assays and (C and E) summary data are shown. Data are means ± SEM from at least eight independent experiments. **P < 0.01 compared with control conditions.

FIG. 3.

Recordings of fura-2 fluorescence in human monocytes. A: OAG-induced calcium influx was measured under control conditions (5.6 mmol/l d-glucose, ○) and in the presence of high glucose (30 mmol/l d-glucose) without (■) and with (▲) TMP (100 μmol/l). B: Summary data showing intracellular calcium levels after treatment of monocytes with high d-glucose for 4 h (control) stimulated with ONOO in the absence and presence of the membrane-permeable TRPC blocker, 2-APB. **P < 0.01.