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Pancreatic β-cell death in type 2 diabetes has been related to p53 subcellular localisation and phosphorylation. However, the mechanisms by which p53 is phosphorylated and its activation in response to oxidative stress remain poorly understood. Therefore, the aim of this study was to investigate mitochondrial p53 phosphorylation, its subcellular localisation and its relationship with apoptotic induction in RINm5F cells cultured under high glucose conditions. Our results show that p53 phosphorylation in the mitochondrial fraction was greater at ser392 than at ser15. This increased phosphorylation correlated with an increase in reactive oxygen species, a decrease in the Bcl-2/Bax ratio, a release of cytochrome c and an increase in the rate of apoptosis. We also observed a decline in ERK 1/2 phosphorylation over time, which is an indicator of cell proliferation. To identify the kinase responsible for phosphorylating p53, p38 mitogen-activated protein kinase (MAPK) activation was analysed. We found that high glucose induced an increase in p38 MAPK phosphorylation in the mitochondria after 24-72 h. Moreover, the phosphorylation of p53 (ser392) by p38 MAPK in mitochondria was confirmed by colocalisation studies with confocal microscopy. The addition of a specific p38 MAPK inhibitor (SB203580) to the culture medium during high glucose treatment blocked p53 mobilisation to the mitochondria and phosphorylation; thus, the release of cytochrome c and the apoptosis rate in RINm5F cells decreased. These results suggest that mitochondrial p53 phosphorylation by p38 MAPK plays an important role in RINm5F cell death under high glucose conditions.

Apoptosis of granulosa cells during follicular atresia is preceded by oxidative stress, partly due to a drop in the antioxidant glutathione (GSH). Under oxidative stress, GSH regeneration is dependent on the adequate supply of NADPH by glucose-6-phosphate dehydrogenase (G6PD). In this study, we analyzed the changes of G6PD, GSH, and oxidative stress of granulosa cells and follicular liquid and its association with apoptosis during atresia of small (4-6 mm) and large (O6 mm) sheep antral follicles. G6PD activity was found to be higher in granulosa cells of healthy small rather than large follicles, with similar GSH concentration in both cases. During atresia, increased apoptosis and protein oxidation, as well as a drop in GSH levels, were observed in follicles of both sizes. Furthermore, the activity of G6PD decreased in atretic small follicles, but not in large ones. GSH decreased and protein oxidation increased in follicular fluid. This was dependent on the degree of atresia, whereas the changes in G6PD activity were based on the type of follicle. The higher G6PD activity in the small follicles could be related to granulosa cell proliferation, follicular growth, and a lower sensitivity to oxidative stress when compared with large follicles. The results also indicate that GSH concentration in atretic follicles depends on other factors in addition to G6PD, such as de novo synthesis or activity of other NADPH-producing enzymes. Finally, lower G6PD activity in large follicles indicating a higher susceptibility to oxidative stress associated to apoptosis progression in follicle atresia.

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The in vitro interaction between purified bovine liver and sperm DNA with somatic histones, to form nucleosomes, and with bovine and salmon protamines were studied. DNAse or microccocal nuclease digestion of liver DNA-histone reassociated chromatin produced the expected polynucleosome type of fragments. Electrophoretic patterns of digested sperm-DNA nucleosomes were different. Micrococcal nuclease digestion produced mainly fragments smaller than 100 bp and some nucleosome-type particles. Under DNAse activity most of the products were smaller than 100 bp, indicating an increased susceptibility of the sperm DNA-histone complexes to the hydrolytic activity of both nucleases, particularly toward DNAse I. This differential susceptibility was confirmed by sucrose gradient spectrophotometric analysis. Acridine orange (AO) staining of histone-DNA reassociated nucleosomes showed significant differences in fluorescence intensity, sperm DNA-histone complexes being almost twice as fluorescent as liver DNA-histone complexes. On the contrary, liver DNA/protamine complexes stained with AO were consistently more fluorescent than sperm DNA-protamine complexes. Finally, no differences in either fluorescence intensity or spectra were observed when liver and sperm DNA were stained with AO after interaction with salmon protamines. The data suggest that sperm DNA has important structural characteristics that differentiates it from somatic DNA. These differences seem to be species specific and must surely play an important role on the determination of the dramatic sequence of that participates sperm chromatin organization.

Glucosamine (GlcN)-induced insulin resistance is associated with an increase in O-linked-N-acetylglucosaminylated modified proteins (O-GlcNAcylated proteins). The role played by O-GlcNAc-selective-N-acetyl-β-D-glucosaminidase (O-GlcNAcase), which removes O-N-acetyl-glucosamine residues from O-GlcNAcylated proteins, has not yet been demonstrated. We investigated whether GlcN-induced whole-body insulin resistance is related to tissue O-GlcNAcase activity and mRNA expression. GlcN (30 µmol/kg/min) or physiological saline (control) was intravenously infused into Sprague-Dawley rats for 2 h. After GlcN treatment, rats were subjected to the following: intravenous glucose tolerance test, insulin tolerance test or removal of the liver, muscle and pancreas. GlcN was found to provoke hyperglycemia compared to control (8.6 ± 0.41 vs. 4.82 ± 0.17 mM, p < 0.001). The insulin resistance index (HOMA-IR) increased (15.76 ± 1.47 vs. 10.14 ± 1.41, p < 0.001) and the β-cell function index (HOMA-β) diminished (182.69 ± 22.37 vs. 592.01 ± 103, p < 0.001). Liver glucose concentration was higher in the GlcN group than in the control group (0.37 ± 0.04 vs. 0.24 ± 0.038 mmol/g dry weight, p < 0.001). Insulin release index (insulin/glucose) was less in the GlcN group than in the control (2.2 ± 0.1 vs. 8 ± 0.8 at 120 min, p < 0.001). In the GlcN group, muscle O-GlcNAcase activity diminished (0.28 ± 0.019 vs. 0.36 ± 0.018 nmol of p-nitrophenyl/mg protein/min, p < 0.001), and K_m increased (1.51 ± 0.11 vs. 1.12 ± 0.1 mM, p < 0.001) compared to the control. In the GlcN group, O-GlcNAcase activity/mRNA expression was altered (0.6 ± 0.07 vs. 1 ± 0.09 of control, p < 0.05). In conclusion, O-GlcNAcase activity is posttranslationally inhibited during GlcN-induced insulin resistance.

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