Fig 2: Enhanced GPx activity compensates for the decreased GPx1 expression in HUVECs treated with high glucose or MG. HUVECs were exposed to D-glucose (Glc, n = 5) or MG (n = 4) for 24 h. D-Mannitol (D-Man) was used as osmotic control for 22 mM D-glucose, MG was scavenged by aminoguanidine (AG, 500 µM). GPx1 protein levels were determined by (A) Western blot analysis (the insert shows exemplary Western blots). (B) GPx enzymatic activity was measured as indicated in the Material and Methods section, and normalized to the GPx1 protein content. (C) For inhibition of the proteasome (exemplary Western blot analysis) cells were pre-incubated for 1 h with 5 nmol/L bortezomib (0.01% in DMSO); therefore a DMSO control was added. *p < 0.05 vs control. Note that the endothelial cell basal medium used as control (contr) itself contained 5.55 mmol/L D-glucose.

Fig 3: Maternal antioxidant enzyme activity and gene expression in the liver at 19dG. (A) SOD activity (U/mg protein); (B) SOD gene expression; (C) SOD inmunostained area (%); (D) Representative micrograph of SOD-1 (40×); (E) GPx activity (mU/mg protein); (F) GPx gene expression; (G) GPx 1/2 inmunostained area (%); (H) Representative micrograph of GPx 1/2 (40×). Values are mean ± SEM, (C: control, n = 8; Cres: control + resveratrol, n = 10; MO: maternal obesity, n = 7; MOres: maternal obesity + resveratrol, n = 7). Within the same group (C vs. Cres and MO vs. MOres) means labelled with different letters differ, p < 0.05; * different in comparison to the respective control (MO vs. C and MOres vs. Cres), p < 0.05. I = interaction between maternal diet and maternal resveratrol intervention, n.s. = not significant.

Fig 4: Carbonylation-associated increase in GPx activity. (A) Specific quantification of GPx1 carbonylation in HUVECs incubated with 22 mmol/L D-glucose (Glc) for 24 h using a sandwich GPx1 ELISA and DNPH-derivatized cell lysates. IgG-antibody (IgG) and a derivatization control solution (contr) were used to demonstrate overall specificity. n = 3, *p < 0.05 vs IgG control. (B) Measuring enzymatic activity of a recombinant GPx1 protein (contr) under mild oxidative conditions in the presence of iron (Fe(II)) and MG. Statistical summary, n = 7–13, *p < 0.05 vs control. Fe (II) metal specificity of hydroperoxide-dependent GPx1 carbonylation was demonstrated using comparable concentrations of Fe(III) and Cu(II) (n = 3). The insert shows recombinant GPx1 in vitro carbonylated under mild (0.05% (w/w) cumene hydroperoxide) or strong (0.3% (w/w) hydrogen peroxide) oxidative conditions and detected by Western blot analysis (exemplary images). Note the aggregated GPx1 protein marked by arrows, which might reflect an oxidative linkage between GPx1 monomers. (C, D) Location of the mutated residues in the 3D structure of human GPx1. (C) Side view of GPx1 homodimers showing selenic acid at the selenocysteine site (yellow, van der Waals spheres representation), the four EKCE amino acids (stick representation), and the intermolecular distances between the C-alpha atoms (dashed lines). The surface representation of the left monomer in the dimeric structure (white) shows the active site located in a shallow depression around the selenic acid. (D) GPx1 dimer interface. The right monomer (gray, cartoon representation) of the dimer structure shown in (C) was rotated by 90° to show the dimer interface. Additionally, the selenic acid is shown in yellow spheres on the monomer along with the intramolecular distances between the corresponding C-alpha atoms for the mutated residues. (E) Effects of different mutations on GPx1 enzymatic activity. GPx activity assay with MCF-7 lysates and adenovirally mediated overexpression of wild-type and mutant GPx1 proteins (Ad.GPx1, wild-type protein; Ad.Mut, EKCE replaced by SAIS; Ad.K114, Ad.E116). Ad.eGFP reporter gene expression and NTC (non-transduced control) were used as controls (statistical summary, n = 4, *p < 0.05 vs NTC). The insert shows an exemplary Western blot of recombinant wild-type and mutant GPx1 proteins, demonstrating the presence of roughly equal quantities of overexpressed wild-type and mutant proteins in MCF-7 cells. ß-Actin served as a loading control.

Fig 5: Oxidative stress biomarkers and antioxidant enzyme activity in male and female liver at 19dG. Male and female: (A,G) MDA concentration (nmol/mg protein); (B,H) ROS concentration (nmol DCF/mg protein/min); (C,I) Carbonyls concentration (μmol PC/mg protein); (D,J) 8-oxo-dG concentration (8-oxo-dG nM/DNA (μg/μL)); (E,K) SOD activity (U/mg protein); (F,L) GPx activity (mU/mg protein). Values are mean ± SEM, (C: control, n = 8; Cres: control + resveratrol, n = 10; MO: maternal obesity, n = 7; MOres: maternal obesity + resveratrol, n = 7). Within the same group (C vs. Cres and MO vs. MOres) means labelled with different letters differ, p < 0.05; * different in comparison to the respective control (MO vs. C and MOres vs. Cres), p < 0.05. I = interaction between maternal diet and maternal resveratrol supplementation, n.s. = not significant.