FIGURE 5.

Effect of glucose energization on glutathione redox in Min6 cells. A, Min6 cells were starved for 1 h and then incubated in 25 or 1 mm glucose in the absence or presence of 10 μm H₂O₂ +1 mm BioGEE. Cells were then lysed in RIPA containing 25 mm N-ethylmaleimide, and BioGEE-tagged proteins were enriched using streptavidin beads. UCP2 was detected by immunoblot. B, HPLC analysis of GSH/GSSG ratio and total GSH associated with proteins in Min6 cells exposed to 25 or 1 mm glucose. Cells were starved, incubated in KRB containing 25 or 1 mm glucose, and then lysed with 0.1% (v/v) trifluoroacetic acid/methanol solution (90:10). Supernatant was then injected into the HPLC. For GSH associated with protein (total glutathione associated with proteome), protein was treated with KOH and then the resulting supernatant was injected into the HPLC. GSH and GSSG retention times were confirmed and quantified by injecting standard solutions. n = 3, mean ± S.E., Student's t test. C, immunodetection of BioGEE-modified proteins in Min6 cell extract. Cells were treated with BioGEE and lysed, and the amount of BioGEE-tagged protein was detected with avidin-HRP. D, UCP2 knockdown increases cellular ROS levels following H₂O₂ challenge. Min6 cells transduced with either short hairpin control (shCtl) or UCP2 (shUCP2) lentiviral particles were loaded with DCFHDA (20 μm) during cell starvation, washed with KRB, and then incubated for 1 h in 25 or 1 mm glucose with H₂O₂ (0–100 μm). Cells were then measured for DCFHDA fluorescence. Data were normalized to total cell protein/well and background fluorescence. n = 4, mean ± S.E., Student's t test. E, time course analysis of ROS production in mitochondria (MitoSOX) or total cell (DCFHDA) following exposure to 25 or 1 mm glucose. Amount of ROS was then detected following various incubation times. Data were normalized to total protein levels. n = 4, mean ± S.E., one-way ANOVA with Fisher's protected least significant difference post hoc test. A.U., absorption units.