Resveratrol attenuates testicular apoptosis in type 1 diabetic mice: Role of Akt-mediated Nrf2 activation and p62-dependent Keap1 degradation

Abstract

Infertility is a common complication in diabetic men, mainly due to the loss of germ cells by apoptotic cell death. However, effective and safe approaches to prevent diabetic induction of testicular apoptosis for diabetic patients have not been available. Resveratrol (RSV), a group of compounds called polyphenols from plants, has been indicated its promising used clinically for cancers and cardiovascular diseases. Therefore, the present study aimed determining whether RSV attenuates type 1 diabetes (T1D)-induced testicular apoptotic cell death in a mouse model. We found that testicular apoptosis and oxidative stress levels were significantly higher in T1D mice than control mice. In addition, the phosphorylation level of metabolism-related Akt and GSK-3β was downregulated and Akt negative regulators PTEN, PTP1B and TRB3 were upregulated in the T1D group. These effects were partially prevented by RSV treatment. Nrf2 and its downstream genes, such as NQO-1, HO-1, SOD, catalase and metallothionein were significantly upregulated by RSV treatment. In addition, RSV-induced Nrf2 activation was found due to Keap1 degradation, mainly reliant on p62 that functions as an adaptor protein during autophagy. These results indicate that the attenuation of T1D-induced testicular oxidative stress and apoptosis by RSV treatment was mainly related to Akt-mediated Nrf2 activation via p62-dependent Keap1 degradation.

Keywords: Apoptosis; Kelch-like ECH-associated protein 1; Nuclear factor erythroid 2-related factor 2; Resveratrol; Testis; Type 1 diabetes; p62.

Graphical abstract

Fig. 1

Effects of RSV on T1D-induced testicular apoptosis. T1D was induced in mice by multiple low-doses of STZ at 50 mg/kg daily for 5 days. After the onset of hyperglycemia, diabetic and age-matched control mice were treated with or without RSV at 20 mg/kg every other day for 4 months. At the end of the treatment period, blood glucose levels (A), testis weight/tibia length ratios (B) were determined. Testicular apoptotic cell death was examined by TUNEL staining. TUNEL-positive cells were quantitatively analyzed (C). Testicular apoptosis expression was examined by western blotting assay for the expression of cleaved-caspase3 (D). Data are presented as mean ± SD (n = 6 at least in each group). *, P < 0.05 vs. control group; #, P < 0.05 vs. DM.

Fig. 2

Effects of RSV on T1D-induced testicular oxidative damage. T1D and control mice were subjected to the same treatment, as described in Fig. 1. Testicular oxidative damage was examined by western blotting assay for the expression of 3-NT as an index of protein nitration (A) and 4-HNE as an index of lipid peroxidation (B). Activity of MDA was confirmed by chemical quantification assay (C). Data are presented as means ± SD (n = 6 at least, in each group). *, P < 0.05 vs. control group; #, P < 0.05 vs. DM.

Fig. 3

Effects of RSV on testicular Nrf2 expression and function. T1D and control mice were subjected to the same treatment, as described in Fig. 1. The expression of Nrf2 was detected by western blotting assay (A), for which the ratio of nuclear Nrf2/cytosolic Nrf2 was presented. mRNA levels of NQO-1, HO-1, SOD, CAT and MT were detected by qRT-PCR (B). Testicular expression of NQO-1 (C), HO-1 (C) and MT (D) were measured by western blotting assay. Activity of SOD (E) and CAT levels (F) were assayed using the corresponding quantification kits. Data are presented as means ± SD (n = 6 at least, in each group). *, P < 0.05 vs. control group; #, P < 0.05 vs. DM.

Fig. 4

Effects of RSV on testicular Akt expression and function. T1D and control mice were subjected to the same treatment, as described in Fig. 1. Testicular expression of phosphorylated and total Akt, GSK-3β and Fyn were measured by western blotting assay (A). The expression of PTEN, TRB3 and PTP1B (negative regulators of Akt) were also measured by western blotting assay (B). Data are presented as means ± SD (n = 6 at least, in each group). *, P < 0.05 vs. control group; #, P < 0.05 vs. DM.

Fig. 5

Effects of RSV on testicular autophagic Keap1 degradation. T1D and control mice were subjected to the same treatment, as described in Fig. 1. Testicular expression of Keap1 (A), p62 (A), and LC3II (B) were measured by western blotting assay. Data are presented as means ± SD (n = 6 at least, in each group). *, P < 0.05 vs. control group; #, P < 0.05 vs. DM.

Fig. 6

Working hypothesis for the mechanism by which RSV protects the testis from diabetes. The working hypothesis for the mechanisms underlying RSV-induced attenuation of testicular apoptosis in T1D mice are illustrated in schematic diagram.