Selective protection of nuclear thioredoxin-1 and glutathione redox systems against oxidation during glucose and glutamine deficiency in human colonic epithelial cells

Abstract

Little is known about the relative sensitivities of antioxidant systems in nuclei, mitochondria, and cytoplasm. The present study examined the oxidation of the thiol-dependent antioxidant systems in these subcellular compartments under conditions of limited energy supply of human colonic epithelial HT-29 cells induced by depletion of glucose (Glc) and glutamine (Gln) from the culture medium. Increased oxidation of dichlorofluoroscein (DCF) indicated an increased level of reactive oxygen species (ROS). Redox Western blot analysis showed oxidation of cytosolic thioredoxin-1 (Trx1) and mitochondrial thioredoxin-2 (Trx2) by 24 h, but little oxidation of nuclear Trx1. The Trx1 substrate, redox factor-1 (Ref-1), was also oxidized in cytosol but was reduced in nuclei. Protein S-glutathionylation (PrSSG), expressed as a ratio of protein thiol (PrSH), was also increased in the cytosol, while nuclear PrSSG/PrSH was not. Taken together, the data show that oxidative stress induced by depletion of Glc and Gln affects the redox states of proteins in the cytoplasm and mitochondria more than those in the nucleus. These results indicate that the nuclear compartment has better protection against oxidative stress than cytoplasm or mitochondria. These results further suggest that energy and/or substrate supply may contribute to sensitivity of mitochondrial and cytoplasmic systems to oxidative damage.

Figure 1

Depletion of Glc, Gln, or Glc and Gln elevates ROS level in cells. DCF fluorescence as a measure of ROS detection was examined in HT29 cells exposed to complete medium (Control), Glc (-Glc), Gln (-Gln), or Glc-and Gln- (-Glc,-Gln) free medium for 1 h. Data represent the mean ± SE of 8 determinations. *P<0.05.

Figure 2

Glc and Gln depletion selectively oxidized cytosolic Trx1 and mitochondrial Trx2 without oxidizing nuclear Trx1. Confluent HT29 cells cultured in complete medium, washed with PBS twice, and then exposed to Glc-, Gln-, or Glc- and Gln-free medium for indicated time. As controls, cells were treated with DTT or H₂O₂ for 30 min. Redox changes in cytosolic (A) and nucleus Trx1 (B), and mitochondrial Trx2 (C) were analyzed by the methods described in previous report [7].

Figure 3

Effect of Glc and Gln depletion on cytosolic and nuclear Ref-1. HT29 cells treated with Glc- and Gln-free medium for indicated time (0, 24, 48 h) were analyzed for reduced form (top) of Ref-1 and total Ref-1 protein amount (middle) after cytosolic and nuclear fractionation. Bottom graph shows % of reduced Ref-1 normalized to total Ref-1 protein.

Figure 4

Effect of Glc and Gln depletion on HT29 cellular GSH, GSSG, and GSH/GSSG redox state (E_h). Cells treated with Glc- and Gln-free medium were analyzed for GSH and GSSG (A, filled circle and square: complete medium, empty circle and square: Glc-and Gln-free medium), and redox state (E_h) for GSH/GSSG (B, filled: complete medium, empty: Glc-and Gln-free medium) was calculated with the Nernst equation.

Figure 5

Effect of Glc and Gln depletion on cytosolic S-glutathionylation (glutathione-protein mixed disulfide; PrSSG, A top), protein thiol content (PrSH, A middle), cytosolic S-glutathionylation/Protein thiol (PrSSG/PrSH, A bottom), nuclear S-glutathionylation (PrSSG, B top), nuclear protein thiol content (PrSH, B middle), and nuclear protein Sglutathionylation/protein thiol (B bottom). As controls, cells treated with complete medium were shown as filled circle (A, cytosolic) and square (B, nucleus).