Diabetes Metab J.  2025 Mar;49(2):210-224. 10.4093/dmj.2024.0099.

Rbbp6-Mediated Bmal1 Ubiquitination Inhibits YAP1 Signaling Pathway to Promote Ferroptosis in Diabetes-Induced Testicular Damage

• Tian Y
• Zhu Z
• Qiao J
• Liu B
• Xiao Y

• KMID: 2565345
• DOI: http://doi.org/10.4093/dmj.2024.0099

Abstract

Background
Diabetes-induced testicular damage (DITD) is a common complication of diabetes. We investigated underlying mechanism of retinoblastoma-binding protein 6 (Rbbp6)-mediated brain and muscle ARNT-like 1 (Bmal1) ubiquitination in modulating ferroptosis in DITD.
Methods
Spermatogenic cell apoptosis and viability were measured by flow cytometry and cell counting kit 8 (CCK-8), respectively. The impact of Rbbp6 and Bmal1 on ferroptosis was assessed by determining expression of ferroptosis markers glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11), and levels of malondialdehyde (MDA), glutathione (GSH), iron, and lipid peroxidation. Co-immunoprecipitation was performed to determine the interaction between Rbbp6 and Bmal1, as well as the ubiquitination level of Bmal1. The expression levels of Rbbp6, Bmal1, Yes-associated protein 1 (YAP1), ferroptosis markers, and testicular steroidogenic enzymes were tested by Western blot.
Results
Bmal1 protein expression was significantly downregulated, while Rbbp6 was upregulated in DITD mouse model and high glucose (HG)-induced GC-1 spg cells. Overexpression of Bmal1 improved testicular injury in diabetic mice, reduced 4-hydroxynonenal (4-HNE), MDA, iron levels, and increased expression levels of GPX4, SLC7A11, GSH, as well as testicular steroidogenic enzymes. Rbbp6 decreased Bmal1 level through promoting its ubiquitination. Meanwhile, Rbbp6 knockdown inhibited the ferroptosis of HG-induced GC-1 spg cells, which were abolished by silencing Bmal1. In addition, knockdown of YAP1 or treatment with ferroptosis inducer erastin blocked the above effects caused by Bmal1 overexpression.
Conclusion
Rbbp6-mediated Bmal1 ubiquitination suppressed YAP1 pathway, promoting ferroptosis in DITD. This study highlighted Rbbp6/Bmal1/YAP1 axis as a potential therapeutic target for mitigating DITD.

Keyword

Bmal1 protein, mouse; Ferroptosis; Rbbp6 protein, mouse; Testicular diseases; Ubiquitination; Yap1 protein, mouse

Figure

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  Fig. 1. The protein level of brain and muscle ARNT-like 1 (Bmal1) was downregulated in testicular tissues of diabetic mice and high glucose (HG)-induced GC-1 spg cells. Diabetic mouse model was established by intraperitoneally injecting with streptozocin (STZ; 45 mg/kg) for 5 successive days. In control group, mice were injected with normal saline. (A) Glucometer detected the blood glucose level of mice. (B, C) H&E staining analyzed the pathological changes of testicular tissues and the number of spermatozoa in each seminiferous tubule. (D) The testosterone level in serum was determined by enzyme-linked immunosorbent assay (ELISA) kit. (E, F, G) The expression levels of Bmal1 in testicular tissues were evaluated by immunohistochemistry, Western blot, and quantitative real-time polymerase chain reaction (qRT-PCR) methods. Cell model was established by culturing GC-1 spg cells in HG medium (30 mM) for 24 hours. (H) The viability of HG-induced GC-1 spg cells was analyzed by cell counting kit 8 (CCK-8) method. (I) The apoptosis of HG-induced GC-1 spg cells was measured by flow cytometry method. (J, K) The expression levels of Bmal1 in HG-induced GC-1 spg cells were measured by Western blot and qRT-PCR. Data are expressed as the mean±standard deviation. PI, propidium iodide. aP<0.05, bP<0.01, and cP<0.001.

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  Fig. 2. Overexpression of brain and muscle ARNT-like 1 (Bmal1) inhibited ferroptosis in high glucose (HG)-induced GC-1 spg cells. Cells were transfected with Bmal1 overexpression plasmid, followed by incubating in HG medium for 24 hours. (A) Bmal1 protein level was detected by Western blot. (B) The viability of HG-induced GC-1 spg cells overexpressed Bmal1 was determined by cell counting kit 8 (CCK-8) method. (C) The apoptosis of HG-induced GC-1 spg cells after overexpressing Bmal1 was detected by flow cytometry method. (D) The expression of glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11) (key proteins involved in ferroptosis) was measured by Western blot. (E, F, G) The levels of malondialdehyde (MDA), iron, and glutathione (GSH) were detected by respective kits. (H) The amount of lipid peroxides was determined by BODIPY 581/591 C11 probe and flow cytometry. (I) Western blot analysis of Yes-associated protein 1 (YAP1) in HG-induced GC-1 spg cells overexpressed Bmal1. Data are expressed as the mean±standard deviation. PI, propidium iodide; ROS, reactive oxygen species. aP<0.05, bP<0.01, and cP<0.001.

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  Fig. 3. Overexpression of brain and muscle ARNT-like 1 (Bmal1) inhibited ferroptosis in testicular tissues of diabetic mice. Streptozocin (STZ)-treated mice were intravenously injected with Bmal1 lentiviral vector or not. (A) Glucometer detected the blood glucose level of diabetic mice overexpressed Bmal1. (B, C) H&E staining analyzed the pathological changes of testicular tissues and the number of spermatozoa in each seminiferous tubule across different experimental groups. (D) The testosterone level in serum of diabetic mice was determined by enzyme-linked immunosorbent assay (ELISA) kit. (E) Western blot analysis of the testicular steroidogenic enzymes steroidogenic acute regulatory protein (StAR), cytochrome P450 family 11 subfamily A member 1 (CYP11A1), and 3β-hydroxysteroid dehydrogenase (3β-HSD) in testicular tissues. (F) Representative immunohistochemical (IHC) images of 4-hydroxynonenal (4-HNE) in testicular tissues of diabetic mice. (G) The expression levels of glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11) in testicular tissues of diabetic mice were measured by Western blot. (H, I, J) The levels of malondialdehyde (MDA), iron, and glutathione (GSH) were detected by respective kits in testicular tissues of diabetic mice. (K) Immunohistochemistry method detected Bmal1 expression in testicular tissues of diabetic mice. (L) Western blot analysis of Yes-associated protein 1 (YAP1) in testicular tissues of diabetic mice. Data are expressed as the mean±standard deviation. aP<0.05, bP<0.01, and cP<0.001.

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  Fig. 4. Retinoblastoma-binding protein 6 (Rbbp6) mediated the ubiquitination modification of brain and muscle ARNT-like 1 (Bmal1). (A) UbiBrowser prediction (http://ubibrowser.ncpsb.org.cn/ubibrowser/) of potential ubiquitin ligases of Bmal1. (B) Co-immunoprecipitation (Co-IP) assay was performed to further verify the interaction between Bmal1 and these ubiquitin ligases. (C, D) Western blot analyses of Rbbp6 expression in testicular tissues of diabetic mice and high glucose (HG)-induced GC-1 spg cells. (E) The protein level of Bmal1 in GC-1 spg cells overexpressed Rbbp6 in the presence of the protein synthesis inhibitor cycloheximide (CHX). (F) Ubiquitination of Bmal1 in GC-1 spg cells overexpressed Rbb6p was measured by Co-IP. Data are expressed as the mean±standard deviation. Rbbp5, retinoblastoma-binding protein 5; Taf5, TATA-box binding protein associated factor 5; Ube3a, ubiquitin protein ligase E3A; Ube4b, ubiquitination factor E4B; IgG, immunoglobulin G; STZ, streptozocin; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; IP, immunoprecipitation; IB, immunoblotting. aP<0.01, bP<0.001.

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  Fig. 5. Knockdown of retinoblastoma-binding protein 6 (Rbbp6) inhibited ferroptosis in high glucose (HG)-induced GC-1 spg cells. GC-1 spg cells were transfected with sh-Rbbp6 plasmid or not, followed by incubating in HG medium for 24 hours. (A, B) Quantitative real-time polymerase chain reaction and Western blot analysis of Rbbp6. (C) Ubiquitination of brain and muscle ARNT-like 1 (Bmal1) in HG-induced cells was measured by co-immunoprecipitation (Co-IP) method after knocking down Rbbp6. (D) The viability of HG-induced GC-1 spg cells silenced Rbbp6 was determined by cell counting kit 8 (CCK-8). (E) The apoptosis of HG-induced GC-1 spg cells was measured by flow cytometry after knocking down Rbbp6. (F) Western blot analysis of glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11) in HG-induced GC-1 spg cells after silencing Rbbp6. (G, H, I) The levels of malondialdehyde (MDA), iron, and glutathione (GSH) in HG-induced GC-1 spg cells after silencing Rbbp6 were detected by respective kits. (J) The amount of lipid peroxides was determined by BODIPY 581/591 C11 probe and flow cytometry. (K) Western blot analysis of Yes-associated protein 1 (YAP1) in HG-induced GC-1 spg cells after silencing Rbbp6. Data are expressed as the mean±standard deviation. NC, negative control; IP, immunoprecipitation; IB, immunoblotting; PI, propidium iodide; ROS, reactive oxygen species. aP<0.05, bP<0.01, and cP<0.001.

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  Fig. 6. The inhibiting effect of brain and muscle ARNT-like 1 (Bmal1) overexpression on ferroptosis in testicular tissues was attenuated by erastin treatment in a dose-dependent manner. Streptozocin (STZ)-treated mice were intravenously injected with Bmal1 lentiviral vector and intraperitoneally injected different concentrations of erastin (10, 20, or, 40 mg/kg). (A) Glucometer detected the blood glucose level of diabetic mice from each group. (B, C) H&E staining analyzed the pathological changes of testicular tissues and the number of spermatozoa in each seminiferous tubule of different experimental groups. (D) The expression levels of glutathione peroxidase 4 (GPX4) and solute carrier family 7 member 11 (SLC7A11) in testicular tissues of diabetic mice were measured by Western blot. (E, F, G) The levels of malondialdehyde (MDA), iron, and glutathione (GSH) were detected by respective kits in testicular tissues of diabetic mice. (H) Immunohistochemistry method detected Bmal1 expression in testicular tissues of diabetic mice. (I) Western blot analysis of Bmal1 and Yes-associated protein 1 (YAP1) in testicular tissues of diabetic mice. Data are expressed as the mean±standard deviation. aP<0.05, bP<0.01, and cP<0.001.

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