GRAPHICAL ABSTRACT

Highlights

• Bmal1 was downregulated in diabetes-induced testicular damage (DITD).

• Overexpression of Bmal1 inhibited ferroptosis in DITD.

• Rbbp6 reduced the Bmal1 expression by mediating its ubiquitination degradation.

• The YAP1 pathway was involved in ferroptosis via the Rbbp6/Bmal1 axis.

INTRODUCTION

Diabetes-induced testicular damage (DITD) is a severe complication in diabetes, leading to sexual and endocrine dysfunction [1,2]. Ferroptosis, an iron-dependent form of cell death with lipid peroxidation and reactive oxygen species (ROS) accumulation [3], is implicated in male reproductive dysfunction, including testicular damage [4]. For instance, cadmium-induced ferroptosis damaged spermatogenesis and testicular development in mice, while ferroptosis inhibitors partially mitigated these effects [5]. Therefore, understanding the relationship between DITD and ferroptosis may provide insights into potential therapeutic intervention.

Brain and muscle ARNT-like 1 (Bmal1) is a circadian clock transcription factor expressed in most tissues [6]. Bmal1 dysfunction has been implicated in various diseases, such as metabolic [7], neurological [8], and reproductive [9] disorders. Notably, male mice with Bmal1 knockout exhibited problematic mating behaviors [10]. Bmal1 knockdown was proved to inhibit the secretion of testosterone [11]. The above evidence suggested Bmal1 as a potential therapeutic target for male reproduction diseases. Interestingly, the induction of ferroptosis mediated by Bmal1/nuclear factor erythroid 2-related factor 2 (Nrf2) axis damaged blood-testis barrier and impaired sperm development [12]. Moreover, overexpression of Bmal1 was indicated to repress RAS-selective lethal 3 (RSL3)-induced ferroptosis in acute myeloid leukemia cells [13]. We therefore hypothesized that Bmal1 might be able to regulate DITD through the regulation of ferroptosis.

Bmal1 is subject to ubiquitination-mediated degradation [14], and several ubiquitin ligases, such as ubiquitin protein ligase E3A (UBE3A) and STIP1 homology and U-box containing protein 1 (STUB1), have been found to regulate Bmal1 expression [15,16]. Herein, the data from UbiBrowser (http://ubibrowser.ncpsb.org.cn/ubibrowser/) identified a series of potential ubiquitin ligases of Bmal1, with retinoblastoma-binding protein 6 (Rbbp6) receiving the highest score. As an E3 ubiquitin ligase, Rbbp6 was suggested to promote the ubiquitination degradation of tumor suppressor p53 by enhancing the interaction between human double minute 2 (Hdm2) and p53 [17]. However, the biological function of Rbbp6 in male reproduction diseases, especially in DITD, remains unclear. Intriguingly, the data from National Center for Biotechnology Information (NCBI) indicated that Rbbp6 was widely expressed in the testis. Therefore, we hypothesized that Rbbp6/Bmal1 axis may play important roles in the pathogenesis of DITD.

Yes-associated protein 1 (YAP1) is a transcriptional co-activator that regulates cell growth, proliferation, and death [18]. YAP1 has been linked to ferroptosis through modulation of ferroptotic genes, such as solute carrier family 7 member 11 (SLC7A11) and glutathione peroxidase 4 (GPX4) [19]. Interestingly, a previous study revealed the positive regulatory role of Bmal1 on YAP1 expression, indicating that Bmal1 promoted the activation of YAP1 pathway [20]. However, the role of Bmal1/YAP1 axis in the regulation of ferroptosis of DITD needs further investigation.

Based on these reports, we hypothesized that Rbbp6 reduced Bmal1 expression through promoting its ubiquitination degradation, which in turn inhibited the activation of YAP1 signaling pathway and then facilitated ferroptosis, leading to DITD. These findings may offer new insights into the molecular mechanisms of DITD.

METHODS

Cell culture and treatment

Mouse spermatogenic GC-1 spg cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen, Carlsbad, CA, USA) containing 10% fetal bovine serum (Gibco, Carlsbad, CA, USA) and 1% penicillin-streptomycin (Invitrogen) at 37°C supplied with 5% CO₂. DITD cell model was established by culturing in high glucose (HG) medium (30 mM) for 24 hours, based on condition optimized in previous studies on DITD [1,21,22]. In addition, cells were treated with ferroptosis inducer erastin (2.5, 5, or 10μM, E7781, Sigma-Aldrich, St. Louis, MO, USA) or cycloheximide (CHX, 20 μg/mL, C7698, Sigma-Aldrich) as needed.

Cell transfection

Short hairpin RNAs (shRNAs) targeted Bmal1, Rbbp6, YAP1 (sh-Bmal1, sh-Rbbp6, sh-YAP1) or scrambled oligonucleotides (sh-NC) were purchased from GenePharma (Shanghai, China). The full length of Rbbp6 coding sequence was amplified and cloned into pcDNA3.1 vector (Invitrogen) to overexpress Rbbp6. GC-1 spg cells were transfected with the above plasmids using Lipofectamine 3000 transfection reagent (Invitrogen). pLenti-CMV-MCS-mPGK-GFP-T2A-Puro lentiviral vector to overexpress Bmal1 was ordered from GenePharma. To package lentivirus, HEK 293T cells were co-transfected with Lenti-Pac HIV Expression Packaging Mix (GeneCopoeia, Guangzhou, China) and the Bmal1 lentiviral vector or the control lentiviral vector using Lipofectamine 3000. Cells infected with the above lentiviral vectors were treated with puromycin (2μg/mL, Sigma-Aldrich) for 2 weeks to select the stably transfected cells.

Co-immunoprecipitation

Co-immunoprecipitation (Co-IP) was utilized to elucidate the interaction between Bmal1 and various E3 ubiquitin ligases (Rbbp6, retinoblastoma-binding protein 5 [Rbbp5], TATA-box binding protein associated factor 5 [Taf5], Ube3a, and ubiquitination factor E4B [Ube4b]), and to determine the impact of Rbbp6 overexpression on the ubiquitinated level of Bmal1. Briefly, cells were collected and lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (P0013B, Beyotime, Shanghai, China) supplemented with protease inhibitor cocktail. Lysates were incubated on ice for 20 minutes and centrifugated at 12,000 g for 25 minutes at 4°C. Next, the supernatant was incubated with anti-Bmal1 antibody (1:100, ab230822, Abcam, Cambridge, UK) which was diluted in phosphate buffer saline containing 0.1% Tween-20 and 1% bovine serum albumin. Normal rabbit immunoglobulin G (1:100, ab172730, Abcam) was used as a negative control under the same condition. After overnight incubation, protein A/G beads were added to capture the antibody-protein complexes and proteins pulled down by the antibodies were purified and detected by Western blot. For the ubiquitination assay, some cell lysates were treated with the proteasome inhibitor MG-132 (Sigma-Aldrich) before immunoprecipitation to prevent degradation of proteins. Subsequently, Western blot analysis was performed using anti-ubiquitin antibody (1:1,000, ab134953, Abcam) to detect ubiquitinated Bmal1.

In vivo mouse model

All mouse-related experiments were approved by the Animal Care and Use Committee of the Affiliated Hospital of Guizhou Medical University, Clinical Medical College of Guizhou Medical University. Male C57BL/6 mice (6 weeks, SLAC Laboratory Animal Center, Shanghai, China) were divided into two groups: control (n=10, mice were injected with normal saline) and streptozocin (STZ, n=60) groups. Mice in the second group were intraperitoneally injected with 45 mg/kg STZ (S0130, Sigma-Aldrich) for 5 successive days to establish diabetic model. The fasting blood glucose levels were measured daily and the diabetic model was successfully established when it was greater than 16.7 mmol/L for 3 days. The blood glucose levels were detected by a glucometer (Roche Diagnostics GmbH, Mannheim, Germany). Subsequently, diabetic mice were further divided into six groups: STZ, STZ+vector, STZ+ Bmal1, STZ+Bmal1+erastin (10 mg/kg), STZ+Bmal1+erastin (20 mg/kg), STZ+Bmal1+erastin (40 mg/kg). Ten mice were included in each group. The STZ+Bmal1 group received intravenous injection of pLenti-CMV-MCS-mPGK-GFP-T2A-Puro lentiviral vector overexpressing Bmal1 (viral titer, 1×10⁹ TU/mL; dose, 200 μL per mouse), while the STZ+ vector group received intravenous injection of the control lentiviral vector (viral titer, 1×10⁹ TU/mL; dose, 200 μL per mouse). Mice were intraperitoneally injected with erastin every 4 days for 4 weeks. After 4 weeks, mice were euthanized with intraperitoneal injection of 120 mg/kg sodium pentobarbital (P-010, Sigma-Aldrich), and testicular tissues and serum samples were collected for subsequent experiments.

Measurement of testosterone level in serum

Supernatant was isolated from blood samples through centrifuging at 4,000 r/min for 10 minutes. Afterwards, testosterone levels in serum were determined by testosterone enzyme-linked immunosorbent assay (ELISA) kit (582701, Cayman Chemical, Ann Arbor, MI, USA). Absorbance was recorded at 405 nm with a microplate reader.

Histopathology analysis on testicular tissues

Testicular tissues were fixed with 4% paraformaldehyde and embedded in paraffin, followed by cutting into 5 μm thickness slices. After staining with hematoxylin and eosin (H&E, ab245880, Abcam), slices fixed in slides were observed at 100× magnification under a microscope (Nikon, Tokyo, Japan). The number of spermatozoa in each seminiferous tubule of different groups was counted.

Immunohistochemistry

Slices of testicular tissues were subjected to deparaffinized, rehydrated, and antigen retrieval by heating in a microwave oven in 10 mmol/L citrate buffer for 3 minutes. Then, sections were incubated overnight at 4°C with primary antibodies against Bmal1 (1:500, ab230822, Abcam) and 4-hydroxynonenal (1:50, 4-hydroxynonenal [4-HNE], ab48506, Abcam). The antibodies were diluted in immunohistochemical staining primary antibody dilution buffer (P0103, Beyotime). For negative control group, section was only incubated with antibody diluent. Afterward, slices were incubated with horseradish peroxidase-conjugated secondary antibodies (1:1,000, 31430 or 31460, Invitrogen) for 2 hours, which diluted in QuickBlock secondary antibody dilution buffer (P0267, Beyotime). Then, the sections were detected using 3,3’-diaminobenzine (Sigma-Aldrich) and counterstained with hematoxylin. The immunohistochemical reaction was visualized under a microscope (Nikon).

Statistical analyses

All experiments were performed in at least three biological replicates, and each biological replicate contained three technical replicates. GraphPad Prism version 8.0 (GraphPad Software Inc., San Diego, CA, USA) was used for statistical analysis. Data are expressed as the mean±standard deviation. Student’s t-test was performed to evaluate the differences between two groups. One-way analysis of variance followed by Tukey post hoc test was performed to determine significant differences between multiple groups. P value <0.05 was considered as statistically significant. More detailed methods were shown in Supplementary Methods.

RESULTS

The protein level of Bmal1 was downregulated in testicular tissues of diabetic mice and HG-induced GC-1 spg cells

A diabetic mouse model was established by STZ injection, as evidenced by elevated blood glucose level (>16.7 mmol/L) (Fig. 1A). Histological analyses revealed separation of seminiferous tubules (Fig. 1B) and the reduced number of spermatozoa in seminiferous tubules (Fig. 1C) from testicular tissues of STZ-treated mice, accompanied by decreased serum testosterone level (Fig. 1D). Fig. 1E and F highlighted that Bmal1 protein levels were notably downregulated in diabetic testicular tissues, and Bmal1 was mainly located in spermatogonia. No significant changes with the mRNA level of Bmal1 were shown in Fig. 1G. On the other hand, GC-1 spg cells were cultured in HG medium to establish in vitro model. The reduced cell viability (Fig. 1H) and increased apoptotic rate (Fig. 1I) were observed, suggesting that cell model was successfully established. Interestingly, Bmal1 protein level in HG-induced GC-1 spg cells also showed downregulation (Fig. 1J), and its gene expression also remained unchanged (Fig. 1K). Taken together, the protein levels of Bmal1 were decreased in injured testicular tissues and HG-induced GC-1 spg cells.

Overexpression of Bmal1 inhibited ferroptosis in HG-induced GC-1 spg cells

To further explore the biological function of Bmal1 in vitro, HG-induced GC-1 spg cells were overexpressed Bmal1 (Fig. 2A). Bmal1 overexpression facilitated the viability of HG-induced GC-1 spg cells (Fig. 2B), while repressing their apoptosis (Fig. 2C). Moreover, Bmal1 overexpression increased the expression levels of ferroptosis regulators GPX4 and SLC7A11 in HG-induced GC-1 spg cells (Fig. 2D). ROS accumulation, lipid peroxidation, glutathione (GSH) depletion and iron levels are critical events in ferroptosis [23]. Bmal1 overexpression reduced malondialdehyde (MDA) (Fig. 2E) and iron (Fig. 2F) levels, while increased GSH level (Fig. 2G) in HG-induced cells. The reduced lipid peroxidation was also observed in HG-induced GC-1 spg cells after overexpressing Bmal1 (Fig. 2H). Mechanistically, overexpression of Bmal1 induced the expression of the YAP1 (Fig. 2I). Thus, Bmal1 overexpression inhibited ferroptosis in HG-induced GC-1 spg cells through activating YAP1 pathway.

Overexpression of Bmal1 inhibited ferroptosis in testicular tissues of diabetic mice

The biological function of Bmal1 in DITD mouse model was also investigated. Bmal1 overexpression reduced blood glucose level (Fig. 3A), improved testicular injury (Fig. 3B), increased the number of spermatozoa in seminiferous tubules (Fig. 3C), and also induced serum testosterone level (Fig. 3D). To clarify whether the changes in serum testosterone level are due to testicular steroidogenesis alteration, the levels of steroidogenic enzymes including steroidogenic acute regulatory protein (StAR), cytochrome P450 family 11 subfamily A member 1 (CYP11A1), and 3β-hydroxysteroid dehydrogenase (3β-HSD) were measured [24]. Bmal1 overexpression partially restored the expression of these enzymes in testicular tissues of STZ-treated mice (Fig. 3E). Bmal1 overexpression decreased 4-HNE, a marker of lipid peroxidation, which primarily localized in the cytoplasm of Sertoli cells (Fig. 3F). Overexpression of Bmal1 also inhibited MDA (Fig. 3H) and iron (Fig. 3I) levels, while promoting GPX4, SLC7A11 (Fig. 3G), and GSH (Fig. 3J) levels in testicular tissues. Furthermore, we observed upregulation of Bmal1 (Fig. 3K) and YAP1 (Fig. 3L) in testicular tissues of DITD mice after overexpressing Bmal1. These findings suggested that Bmal1 overexpression inhibited ferroptosis via activating the YAP1 pathway in the DITD mouse model.

Rbbp6 mediated the ubiquitination modification of Bmal1

Next, UbiBrowser database identified several potential ubiquitin ligases of Bmal1, including Rbbp6, Rbbp5, Taf5, Ube3a, and Ube4b (Fig. 4A). Through Co-IP assay, Rbbp6 was found to be most clearly co-immunoprecipitated with Bmal1 protein (Fig. 4B). Subsequently, Rbbp6 was upregulated both in testicular tissues of DITD mice (Fig. 4C) and HG-induced GC-1 spg cells (Fig. 4D). Also, Bmal1 protein levels were dramatically decreased in cells overexpressed Rbbp6 after the protein synthesis inhibitor CHX treatment (Fig. 4E). The increased ubiquitination modification of Bmal1 was found in cells treated with the proteasome inhibitor MG-132 and transfected with Rbbp6 plasmid (Fig. 4F), indicating that Rbbp6 regulated the ubiquitination status of Bmal1. These findings indicated that Rbbp6-mediated ubiquitin-proteasome pathway was responsible for Bmal1 degradation.

Knockdown of Rbbp6 inhibited ferroptosis in HG-induced GC-1 spg cells

Next, we began to explore whether Rbbp6 was involved in ferroptosis in HG-induced GC-1 spg cells. Rbbp6 were significantly upregulated in HG-induced GC-1 spg cells, and downregulated after transfecting with sh-Rbbp6 vector (Fig. 5A and B). The ubiquitinated level of Bmal1 was increased in HG-induced GC-1 spg cells, while Rbbp6 knockdown inhibited its ubiquitination degradation (Fig. 5C). Furthermore, Rbbp6 knockdown promoted the viability of HG-induced GC-1 spg cells (Fig. 5D), while repressing their apoptosis (Fig. 5E). Rbbp6 knockdown also promoted GPX4, SLC7A11 (Fig. 5F), and GSH (Fig. 5I) levels, while reduced MDA (Fig. 5G), iron (Fig. 5H), and lipid peroxidation (Fig. 5J) levels in HG-induced GC-1 spg cells, suggesting its inhibitory effect on ferroptosis. In addition, Rbbp6 knockdown increased YAP1 expression in HG-induced GC-1 spg cells (Fig. 5K). These findings indicated the regulatory function of Rbbp6 on ferroptosis of GC-1 spg cells induced by HG.

Rbbp6 regulated ferroptosis in HG-induced GC-1 spg cells by Bmal1

To investigate the regulatory role of the Rbbp6/Bmal1 axis on ferroptosis, rescued assays were performed in HG-induced GC-1 spg cells. The upregulation of Bmal1 and YAP1 caused by silencing Rbbp6 was blocked by Bmal1 knockdown (Supplementary Fig. 1A). Subsequently, the promoting effect on cell viability (Supplementary Fig. 1B) and inhibitory effect on cell apoptosis (Supplementary Fig. 1C) induced by Rbbp6 knockdown were reversed by knocking down Bmal1. Through measuring the expression of GPX4 and SLC7A11, and content of MDA, iron, GSH, and lipid peroxidation, Bmal1 knockdown was observed to block the inhibition of ferroptosis caused by silencing Rbbp6 in HG-induced GC-1 spg cells (Supplementary Fig. 1D-H). Collectively, Rbbp6 induced ferroptosis in HG-induced GC-1 spg cells through mediating ubiquitination degradation of Bmal1.

Bmal1 overexpression inhibited ferroptosis in HG-induced GC-1 spg cells by activating YAP1 signaling pathway

We then began to verify the function of Bmal1/YAP1 axis on cell ferroptosis. We observed that the increased YAP1, GPX4, and SLC7A11 expression caused by overexpressing Bmal1 in HG-induced GC-1 spg cells were reversed by silencing YAP1 or treating with different concentrations of ferroptosis inducer erastin (Supplementary Fig. 2A). The inhibitory effect on these proteins was more pronounced as the concentration of erastin increased (Supplementary Fig. 2A). However, the expression of Bmal1 was not significant changed after knocking down YAP1 or treating with erastin (Supplementary Fig. 2A). YAP1 knockdown or erastin treatment also blocked Bmal1 overexpression-mediated promotion of cell viability (Supplementary Fig. 2B) and inhibition of apoptosis (Supplementary Fig. 2C), with more evident alteration at higher erastin doses. Furthermore, YAP1 knockdown or erastin treatment increased the levels of MDA (Supplementary Fig. 2D) and iron (Supplementary Fig. 2E) and promoted lipid peroxidation (Supplementary Fig. 2G) in cells overexpressed Bmal1, while reducing GSH level (Supplementary Fig. 2F). These effects were also amplified by higher erastin concentrations (Supplementary Fig. 2D-G). In conclusion, Bmal1 inhibited the ferroptosis of HG-induced GC-1 spg cells by activating YAP1 pathway.

The inhibiting effect of Bmal1 overexpression on ferroptosis in testicular tissues was attenuated by Erastin treatment in a dose-dependent manner

We then began to explore whether erastin treatment could repress the effect of Bmal1 overexpression on ferroptosis in vivo. Bmal1 overexpression attenuated the STZ-induced increase in blood glucose, but this effect was reduced by treatment with erastin in a dose-dependent manner (Fig. 6A). Similarly, erastin treatment diminished the protective effect of Bmal1 overexpression on testicular injury (Fig. 6B) and decreased the number of spermatozoa in seminiferous tubules (Fig. 6C) in a dose-dependent manner. Moreover, the increased expression of GPX4 and SLC7A11 in the testicular tissues induced by Bmal1 overexpression was reversed by erastin treatment (Fig. 6D). The reduced MDA (Fig. 6E) and iron (Fig. 6F) levels, and increased GSH level (Fig. 6G) in the Bmal1 overexpression group were dose-dependently reversed by erastin treatment. Additionally, the elevated Bmal1 expression in the testicular tissues of the STZ+Bmal1 group was not obviously altered by erastin treatment (Fig. 6H and I). However, the increased YAP1 expression caused by Bmal1 overexpression in the STZ group was suppressed by erastin treatment in a dose-dependent manner (Fig. 6I). In conclusion, Bmal1 inhibited ferroptosis in the testicular tissues of DITD mice, and this protective effect was attenuated by erastin in a dose-dependent manner.

DISCUSSION

Prolonged hyperglycemia in diabetes leads to testicular degeneration and dysfunction, affecting sperm development and maturation, resulting in infertility [25]. Studies have shown that mammalian sperm cells are more susceptible to oxidative damage [26]. Since oxidative stress-induced lipid peroxidation increases ferroptosis [27,28], the researchers naturally speculated the possibility of regulating ferroptosis on sperm cells in DITD. This study revealed that the downregulation of Bmal1 caused by Rbbp6-mediated ubiquitination modification inhibited YAP1 pathway, thus promoting ferroptosis in DITD.

The disruption of circadian rhythms is linked to male reproductive dysfunction [29,30]. The inhibition of clock gene expression, including Bmal1, might reduce sperm motility and reproductive testosterone secretion [9,11]. Alvarez et al. [31] provided clear evidence that mice lacking Bmal1 experienced the decreased testosterone level, mating difficulties, and infertility. The downregulation of Bmal1 in DITD mouse model and HG-induced GC-1 spg cells was also observed in our study. Interestingly, Bmal1 overexpression ameliorated blood glucose level, testicular injury, and low spermatozoa counts in diabetic mice. More importantly, it partially restored the expression of testicular steroidogenic enzymes including StAR, CYP11A1, and 3β-HSD [24]. These findings highlighted the indispensable roles of Bmal1 in maintaining male reproductive function. Furthermore, Bmal1 overexpression repressed ferroptosis, a non-apoptotic type cell death associated with iron metabolism and lipid peroxidation [32]. Increasing reports revealed that ferroptosis occurred during numerous organ injuries, including reproductive organs [4,33]. Recently, researchers have explored whether Bmal1-regulated ferroptosis plays a role in other diseases, as autophagic degradation of Bmal1 is believed to promote the ferroptosis process in tumors [34]. For instance, Zhang et al. [12] indicated the promoting role on ferroptosis mediated by Bmal1/Nrf2 axis destroyed blood-testis barrier with nicotine treatment. Herein, our findings revealed for the first time that Bmal1 overexpression inhibited ferroptosis in HG-induced GC-1 spg cells and testicular tissues of diabetic mice.

Ubiquitination is one of the most important post-translational modifications. E3 ligases are responsible for recognizing specific substrates and transferring activated ubiquitin molecules to the substrates [35]. The ubiquitination and degradation of Bmal1 was modulated by various enzymes in the ubiquitin-proteasome system [36,37]. For instance, E3 ubiquitin ligase STUB1 suppressed Bmal1 expression by ubiquitin-proteasome pathway, alleviating hydrogen peroxide-induced cell senescence [16]. Herein, E3 ligase Rbbp6 was found to interact with Bmal1 and mediate its ubiquitination degradation. Importantly, we observed upregulation of Rbbp6 in HG-induced GC-1 spg cells and testicular tissues of DITD mice, and Rbbp6 knockdown repressed ferroptosis, which was reversed by silencing Bmal1. Previous studies usually emphasized that Rbbp6 negatively regulated the tumor suppressors p53 and Rb [38]. Our findings for the first time indicated that Rbbp6 acted upstream E3 ubiquitin ligase of Bmal1 in hyperglycemic GC-1 spg cells and DITD mice, regulating cellular ferroptosis.

YAP1 pathway has been suggested be involved in ferroptosis in human diseases and organ injuries. For example, YAP1 inhibited ferritinophagy-mediated ferroptosis in hepatocytes, and YAP1 deficiency aggravated sepsis-induced liver injury [39]. Additionally, YAP1 regulated ferroptosis through SKP2, supporting the connection between Hippo pathway and ferroptosis [40]. These publications suggested that YAP1 regulated multiple aspects of cellular iron metabolism, antioxidant defense, and lipid peroxidation, thereby influencing the susceptibility to ferroptosis. Intriguingly, Bmal1 overexpression promoted phonotypic switch of vascular smooth muscle cells towards fibroblast-like cells through increasing YAP1 level [20]. This is consistent with our findings that Bmal1 contributed to YAP1 pathway activation. Furthermore, YAP1 knockdown was found to reverse the inhibitory effect of Bmal1 overexpression on the ferroptosis of HG-induced GC-1 spg cells. To the best of our knowledge, this is the first observation that demonstrated Bmal1 regulated ferroptosis through the activation of YAP1 pathway.

The present study aimed to investigate the role of the Rbbp6/Bmal1/YAP1 axis on ferroptosis in DITD. We believe that validating the importance of the Rbbp6/Bmal1/YAP1 axis in animal model will provide a more comprehensive understanding of the observed molecular interactions. However, due to time and resource constraints, we can only include some relevant in vivo experiments at present. Additionally, we will focus on investigating the reasons for upregulation of Rbbp6 in DITD model as well as its mechanism in our future research. Moreover, although this study provided insights into Bmal1’s role in DITD, we acknowledge the limitations in exploring its broader effects on circadian rhythms due to our current focus on DITD and fund constraints. Future studies equipped with the necessary technology could further elucidate Bmal1’s systemic impacts, particularly on circadian biology.

In conclusion, our findings may suggest a possible mechanism by which the downregulation of Bmal1 caused by Rbbp6-mediated ubiquitin-proteasome pathway repressed YAP1 expression, promoting ferroptosis, ultimately aggravating DITD. These findings have gone some way towards enhancing our understanding of DITD pathogenesis and might be of assistance to develop novel strategies for DITD treatment.

SUPPLEMENTARY MATERIALS

NOTES

CONFLICTS OF INTEREST

No potential conflict of interest relevant to this article was reported.

AUTHOR CONTRIBUTIONS

Conception or design: Y.X., Z.Z., J.Q., Y.T.

Acquisition, analysis, or interpretation of data: all authors.

Drafting the work or revising: all authors.

Final approval of the manuscript: all authors.

FUNDING

This work was supported by Guizhou Provincial Science and Technology Projects-ZK (2024) 215; National Natural Science Foundation of China (82460291); Guizhou Science and Technology Plan Project Qiankehe Support (2020) No. 4Y142; Guizhou Urology Postgraduate Workstation Guizhou Teaching and Research Institute GZZ word (2016) 04; Guizhou Provincial Health and Health Commission Science and Technology Fund Project GZWJKJ 2018-1-037; Gyfynsfc2021-23; National Natural Science Foundation Cultivation Project of Affiliated Hospital of Guizhou Medical University, Dr. Gyfybsky-2022-8; Affiliated Hospital of Guizhou Medical University; Guizhou Science and Technology Fund Project (Qian Kehe Foundation [2020] 1Y309); Guizhou Provincial Health and Health Commission Science and Technology Fund Project GZWJKJ 2015-1-036; Guizhou Provincial Health and Health Commission Science and Technology Fund Project gzwkj 2021-490.

Acknowledgements

None

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-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.

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.

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.

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.

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.

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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Tian Y, Zhu Z, Qiao J, Liu B, Xiao Y. Rbbp6-Mediated Bmal1 Ubiquitination Inhibits YAP1 Signaling Pathway to Promote Ferroptosis in Diabetes-Induced Testicular Damage. Diabetes Metab J. 2024 Nov 6. doi: 10.4093/dmj.2024.0099. Epub ahead of print.

Received: Mar 01, 2024; Accepted: Jun 17, 2024

DOI: https://doi.org/10.4093/dmj.2024.0099.

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