The protective effect of FGF21 on diabetes-induced male germ cell apoptosis is associated with up-regulated testicular AKT and AMPK/Sirt1/PGC-1α signaling

Abstract

Fibroblast growth factor 21 (FGF21) is a metabolic regulator that is required for normal spermatogenesis and protects against diabetes-induced germ cell apoptosis. Here, we tried to define whether diabetes-induced germ cell apoptosis that is predominantly due to increased oxidative stress was associated with impaired glucose and fatty acid metabolism, by examining the effects of Fgf21 gene knockout (FGF21-KO) or FGF21 treatment on the glucose and fatty acid metabolic pathways in streptozotocin-induced diabetic mice. Western blottings revealed that protein kinase B (AKT)-mediated glucose signaling was down-regulated in diabetic testes and further decreased in FGF21-KO diabetic group both 10 days and 2 months after diabetes onset, reflected by reduced glycogen synthase (GS) kinase (GSK)-3β phosphorylation and increased GS phosphorylation. Deletion of the Fgf21 gene also inactivated fatty acid metabolism-related factors, AMP-activated protein kinase (AMPK), sirtuin 1 (Sirt1), and peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α), along with exacerbating diabetes-induced testicular oxidative stress and damage. Treatment with recombinant FGF21 partially prevented these diabetic effects. In FGF21-KO nondiabetic mice, testicular AMPK/Sirt1/PGC-1α signaling was down-regulated and AKT1 and murine double minute 2 were inactivated along with the increased p53 expression but not AKT2, GSK-3β, and GS. These results suggest that the role of FGF21 in maintaining spermatogenesis is associated with its activation of AKT1 and inhibition of p53. Deletion of the Fgf21gene significantly exacerbates diabetes-induced down-regulation of testicular AKT/GSK-3β/GS and AMPK/Sirt1/PGC-1α pathways and testicular oxidative stress and cell apoptosis.

Figure 1.

Deletion of the Fgf21 gene increases diabetes-induced oxidative damage in the testis. Type 1 diabetes was induced with STZ (200 mg/kg ip). Diabetic and age-matched control mice were administered daily ip injections of FGF21 (100 μg/kg) or PBS for 10 days. To measure the oxidative damage in testis, immunofluorescence staining (A) was done with anti-3-NT antibody and anti-4-HNE antibody, and the nuclear staining was done with DAPI (blue) on testis tissue sections (400×). Scale bar, 50 μm. Expression of 3-NT (B) and 4-HNE (C) was also detected by Western blot analysis, for which multiple bands were grouped as one for the quantitative densitometry analysis so that the results presented in the figures were the combined results. Data are presented as mean ± SD (n = 8). *, P < .05 vs WT-CON; #, P < .05 vs WT-DM; +, P < .05 vs KO-CON; &, P < .05 vs KO-DM.

Figure 2.

Deletion of the Fgf21 gene increases the deficiency of glucose metabolism-associated pathway, AKT/GSK-3β/GS in testes of diabetic mice. Testicular tissue was used for Western blot analysis of phosphorylated and total AKT (A), GSK-3β (B), and GS (C). Data are presented as mean ± SD (n = 8). *, P < .05 vs WT-CON; #, P < .05 vs WT-DM; +, P < .05 vs KO-CON; &, P < .05 vs KO-DM.

Figure 3.

Effects of diabetes and the Fgf21 gene deletion on AKT1 and AKT2 in testis. Phosphorylated and total AKT1 (A) and AKT2 (B) were examined by Western blot analysis. Data are presented as mean ± SD (n = 8). *, P < .05 vs WT-CON; #, P < .05 vs WT-DM; +, P < .05 vs KO-CON; &, P < .05 vs KO-DM.

Figure 4.

Effects of diabetes and the Fgf21 gene deletion on AMPK/Sirt1/PGC-1α pathway associated with energy metabolism. Western blot analyses were used to detect the expression of phosphorylated and total AMPK, Sirt1 and PGC-1α (A) as well as LKB1 and ERK1/2 (B). Data are presented as mean ± SD (n = 8). *, P < .05 vs WT-CON; #, P < .05 vs WT-DM; +, P < .05 vs KO-CON; &, P < .05 vs KO-DM.

Figure 5.

Immunofluorescence staining for Sirt1. A, Immunofluorescence staining using anti-Sirt1 antibody (5), anti-β-actin antibody (green), and DAPI for nuclei (blue) on testicular tissues (400×). Scale bar, 25 μm. B, Percentage of nuclei that was positively stained for Sirt1. Data are presented as mean ± SD (n = 8). *, P < .05 vs WT-CON; #, P < .05 vs WT-DM; +, P < .05 vs KO-CON; &, P < .05 vs KO-DM.

Figure 6.

Effects of diabetes and the Fgf21 gene deletion on p53 and cell apoptosis associated protein. A, Immunohistochemistry staining using antiphosphorylated p53 antibody (400×). Scale bar, 50 μm. B, Western blot analysis was used to quantify p53 activation. C, Western blot analysis to quantify MDM2 activation. Data are presented as mean ± SD (n = 8). *, P < .05 vs WT-CON; #, P < .05 vs WT-DM; +, P < .05 vs KO-CON; &, P < .05 vs KO-DM.

Figure 7.

Chronic effects of diabetes and the Fgf21 gene deletion on testicular apoptosis and associated cell signaling components. Animals were killed at 2 months after diabetes onset. A and B, Apoptotic cell death was examined with TUNEL staining (A) and Western blot analysis for AIF (B). C–F, Western blots were also used to detect the phosphorylated and total expression of AKT1 (C), AKT2 (D), AMPK (E), and p53 (F). Data are presented as mean ± SD (n = 4–5). *, P < .05 vs WT-CON; #, P < .05 vs WT-DM; +, P < .05 vs KO-CON.

Figure 8.

Illustration of the working mechanisms for FGF21 prevention of spontaneous and diabetes-induced germ cell apoptosis. Diabetes adversely affects glucose metabolism (AKT/GSK-3β/GS signaling) and fatty acid oxidation (AMPK/Sirt1/PGC-1α) in testis, leading to the accumulation of metabolic intermediates that cause testicular oxidative damage and germ cell apoptosis. FGF21 deficiency exacerbates these defects, leading to testicular cell apoptosis. In nondiabetics, FGF21 preserves almost normal spermatogenesis through the AKT-signaling pathway to inhibit p53 via AKT1/MDM2.