PMC

HG effects on endogenous antioxidants in C3H and Hcb-19 (TxNIP-deficient) MC. C3H and Hcb-19 MC were cultured in NG (5.6 mm) or HG (25 mm) for 3 and 24 h. Immunoblotting was performed on total cell lysates for: A, GPx1; B, HO-1; C, MnSOD; D, catalase; E, Trx1; and F, Trx2, whereas β-actin served as loading control. The results were normalized to C3H NG, and depicted in the graphs below the respective images. Results are mean ± S.E. (n = 4). *, p < 0.05 versus C3H NG; **, p < 0.001 versus C3H NG; $, p < 0.05 versus C3H 3-h HG; #, p < 0.01 versus C3H 24-h HG; and ##, p < 0.001 versus C3H 3- and 24-h HG.

Effect of HG on isoforms and subunits of NADPH oxidase in MC. Growth-arrested MC from C3H and Hcb-19 mice were cultured in NG (5.6 mm) or HG (25 mm) for 3 and 24 h. Total cell lysates were subjected to immunoblotting for: A, Nox1; B, p47^phox; C, p67^phox; D, rac1; and E, p22^phox, with β-actin as loading control. The results were normalized to C3H NG, and are represented in graphs below the respective images. Results are mean ± S.E. (n = 3–4 independent experiments). *, p < 0.05 versus control C3H NG; #, p < 0.05 versus C3H 3-h HG.

High glucose augments NADPH oxidase activity and Nox4 expression in C3H but not in Hcb-19 MC. A, NADPH oxidase activity in the total cell lysates isolated from NG (5.6 mm) or HG (25 mm)-treated MC was measured as NADPH-dependent superoxide generation with the lucigenin-enhanced chemiluminescence method (n = 4). B and C, expression of Nox2 and Nox4 protein in total cell lysates was assessed by immunoblotting (n = 4). D and E, Nox2 and Nox4 mRNA expression was assessed by real-time PCR (n = 6–7). Values are mean ± S.E. *, p < 0.01 versus C3H NG; #, p < 0.05 versus C3H 3-h HG; and ###, p < 0.0001 versus C3H 3-h HG.

HG-stimulated collagen accumulation is impaired in Hcb-19 TxNIP-deficient MC. Growth-arrested MC (C3H, Hcb-19) were exposed to NG (5.6 mm) or HG (25 mm) for up to 24 h. A, proteins from total cell lysates were separated on SDS-PAGE and blotted for TxNIP and β-actin as loading control. B, collagen IV expression was analyzed by confocal microscopy and quantified by measuring pixel intensity per cell (n = 150 cells) in 3 independent experiments. Results (mean ± S.E.) are shown in the graphs. ***, p < 0.0001 versus C3H NG; **, <p < 0.001 versus C3H NG; and #, p < 0.05 versus C3H HG.

TxNIP-deficient Hcb-19 MC show impaired mitochondrial glucose metabolism and increased lactate production. Growth arrested MC (C3H, Hcb-19) were exposed to NG (5.6 mm) or HG (25 mm) for up to 24 h. A, lactate concentrations in the cell culture media. Results are mean ± S.E. (n = 4), *, p < 0.05 versus C3H NG; and #, p < 0.05 versus Hcb-19 HG. B, MC fluorescence was observed by confocal microscopy after JC-1 treatment for 30 min. The images were analyzed using ImageJ software and normalized to C3H NG. Quantification is illustrated in the graph. Results are mean ± S.E. (n = 3). **, p < 0.001 versus C3H NG; and ##, p < 0.001 versus C3H 3- and 24-h HG.

Overexpression of TxNIP in Hcb-19 MC restores mitochondrial ROS and Nox4 expression. Hcb-19 cells were transduced with GFP (AdGFP) or TxNIP (AdTxNIP) adenoviruses and C3H cells with AdGFP alone to serve as a control. After 24 h, cells were growth arrested for 48 h and then exposed to NG (5.6 mm) or HG (25 mm) for 3 h. A, mitochondrial superoxide production in the MC was determined with MitoSOX dye by confocal microscopy. Quantification is illustrated in the graph. Results are mean ± S.E. *, p < 0.05; and **, p < 0.001 versus AdGFP C3H NG. B, immunoblotting for Nox4 and TxNIP was performed using lysates from Hcb19 MC transduced with varying concentrations of AdTXNIP (25 × 10⁷, 5 × 10⁸, and 1 × 10⁹ ifu/ml) in NG or HG. Results are mean ± S.E. (n = 4). *, p < 0.05 versus AdGFP Hcb19 NG.

Mitochondrial ROS and Nox4 protein expression in MC requires TxNIP. C3H and Hcb-19 MC were treated with NG (5.6 mm) or HG (25 mm) for the times indicated. A, ROS levels were determined by confocal microscopy after MitoSOX treatment for 30 min. The images were analyzed using ImageJ software and normalized to C3H NG designated as 100%. B, mitochondrial and cytosolic extracts were obtained from the total cell lysates and immunoblotted for the mitochondrial markers, prohibitin, VDAC, and COX IV as described under ”Materials and Methods.“ C, mitochondrial expression of Nox4 and prohibitin (mitochondrial loading control) were assessed by immunoblotting. Results are mean ± S.E. (n = 3). *, p < 0.05 versus control C3H NG; **, p < 0.001 versus C3H NG; ##, p < 0.05 versus C3H HG; and ##, p < 0.001 versus C3H HG.

TxNIP knockdown in C3H MC mimics the defects in response to HG observed in Hcb-19 MC. C3H MC were transfected with 50 nm TxNIP-specific siRNA (siTxNIP) or universal negative control siRNA (scrambled, scr) for 24 h and then incubated in NG (5.6 mm) or HG (25 mm) for 3 or 24 h. A and B, intracellular ROS and mitochondrial superoxide formation were assessed by confocal microscopy using DCF and MitoSOX, respectively (n = 3). D, Nox4 expression was measured in total cell lysates of the transfected cells by immunoblotting. Values are mean ± S.E. (n = 3). *, p < 0.05 versus scr siRNA NG; ***, p < 0.001 versus scr siRNA; #, p < 0.05 versus scr HG; and ###, p < 0.001 versus scr HG. E, collagen IV expression was analyzed by confocal microscopy and quantified by measuring pixel intensity per cell (150 cells) in 3 independent experiments. Results are mean ± S.E. (n = 3) depicted in the graphs. C, NADPH oxidase activity in the total cell lysates isolated from NG (5.6 mm) or HG (25 mm)- treated MC, transfected with scrambled siRNA or siTxNIP, was measured as NADPH-dependent superoxide generation with the lucigenin-enhanced chemiluminescence method (n = 4).

High glucose-induced ROS and concomitant decrease in Trx activity is impaired in TxNIP-deficient Hcb-19 MC. MCs were exposed to NG (5.6 mm) or HG (25 mm) for up to 24 h. A-C, MC were assessed by confocal microscopy after DCF or DHE treatments for 30 min. The images were quantified as pixel intensity and expressed as % of C3H NG designated as 100 (n = 3 independent experiments). D, H₂O₂ released by the cells was measured in 50 μl of cell culture media preincubated with Amplex Red reagent for 30 min (n = 6; results were normalized for the number of cells). E, after cell lysis, Trx activity (n = 5) was performed as described under ”Materials and Methods.“ Results are expressed as mean ± S.E. *, p < 0.01 versus C3H NG; **, p < 0.001 versus C3H NG; #, p < 0.05 versus C3H 1-h HG; ##, p < 0.001 versus C3H 3- or 24-h HG; and ###, p < 0.0001 versus C3H 3-h HG. F, C3H and Hcb-19 MC were pretreated with 10 μm diphenyleneiodonium chloride for 1 h and then exposed to 1 and 10 μm H₂O₂ for a total of 4 h. The decline in ROS was assessed between 2 and 3 h post-treatment (see inset for slopes) by DCF fluorescence microscopy and the relative (%) decreases in fluorescence intensities expressed as the rate of disappearance/min. Results are mean ± S.E. from 4 independent experiments. *, p < 0.05 versus C3H at 1 or 10 μm concentrations.