INS-1 cells were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS), 11.1 mM D-glucose, 100 U/mL penicillin, 100 μg/mL streptomycin, 10 mM HEPES, 2 mM L-glutamine, 1 mM sodium pyruvate and 50 µM 2-mercaptoethanol (All from ThermoFisher) as described previously (Wang et al., 2023). For Klotho-treated culture, INS-1 cells were cultured in RPMI 1640 medium supplemented with 10% FBS and subjected to Klotho (1 μM/2 μM, 5334-KL-025, RD systems) treatment in the absence or presence of 30 nM doxorubicin (MCE, MedChemExpress) for 16 h. The soluble recombinant protein (s-KL) we used consists of both KL1 and KL2 domains.

Cells were seeded in 24-well culture plates and treated with 1 μM or 2 μM kL in the presence of 30 nM doxorubicin. Cells were washed with PBS and fixed in SA-β-galactosidase (SA-β-gal) staining solution for 15 min at room temperature. Then, the cells were washed three times with PBS and incubated with SA-β-gal staining solution (Senescence β-Galactosidase Staining Kit, Cell Signaling Technology) for 16–20 h at 37°C. For 24-well plates, 500 µL SA-β-gal staining solution was added to each well. After overnight incubation, the cells were washed with PBS and observed under a bright field microscope.

For INS-1 cell immunofluorescence staining, INS-1 cells were plated onto cover slides. After treatment with doxorubicin in the absence or presence of KL, INS-1 cells were washed with PBS, fixed and permeabilized with 4% paraformaldehyde for 15 min and gently washed with PBS. Cells were blocked with blocking solution (10% normal goat serum, 0.1% Triton X-100 in PBS) for 1 h at room temperature, and rabbit anti-Ki67 (1:100, Thermo Fisher), rabbit anti-P53 (1:100, Cell Signaling Technology), rabbit anti-P21 (1:100, Cell Signaling Technology), rabbit anti-γ-H2AX (1:100, Cell Signaling Technology) and rabbit anti-Klotho (1:200; Cell Signaling Technology) were added overnight at 4°C. The cells were washed three times with PBS for 5 min, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488 (1:1000; ab150077, Abcam) and DAPI (1 μg/mL, 4083S, Cell Signaling Technology). All immunofluorescent images were captured by an Olympus upright BX50 fluorescence microscope (Olympus, Huashan Hospital, Shanghai, China) at ×40 magnification.

RNA isolation and reverse transcription from 1 μg RNA was performed using TRI Reagent (Sigma Aldrich) and reverse transcriptase (Invitrogen, MA, United States), respectively, according to the manufacturer’s instructions. Real-time qPCR reaction was conducted on (Applied biosystems, 7,500 Real Time PCR System, CA, United States) in 10 μL volume of SYBR green PCR reagents (Thermo scientific, MA, United States) under conditions recommended by the supplier. For IGF-1R, P53, P21, TNF-α and GAPDH mRNA detection, the following primers were used: 5’CGG CAG AGC AAA GGA GAC ATA3’ (IGF-1R forward), 5’TGG TGG AGG TGA AAC GGA 3’ (IGF-1R reverse); 5’ACC AGC ACA AGC TCC TCT CC3’ (P53 forward), 5’AAG GCC CTC ATT CAG CTC TCG3’ (P53 reverse); 5’GGC AAG AGT GCC TTG ACG AT3’ (P21 forward), 5’CCT CTT GAC CTG CTG TGT CG3’ (P21 reverse); 5’GAC​GTG​GAA​CTG​GCA​GAA​GAG3’ (TNF-α forward), 5’ TTG​GTG​GTT​TGT​GAG​TGT​GAG3’ (TNF-α reverse); 5’AAC​TTT​GGC​ATT​GTG​GAA​GG3’ (GAPDH forward) and 5’ACA​CAT​TGG​GGG​TAG​GAA​CA3’ (GAPDH reverse). Results were normalized to GAPDH and relative quantification analysis was performed using the 2^−ΔΔCt method.

Protein was extracted from INS-1 cells using radioimmune precipitation assay lysis buffer (Beyotime Biotechnology., Ltd.) supplemented with protease and phosphatase inhibitors (Millipore). The total protein concentration was determined using a Pierce BCA Protein Assay kit (Thermo Scientific). Equal amounts of protein (10 µg) were separated by 12% SDS‒PAGE, transferred onto PVDF membranes, and immunoblotted with rabbit anti-Klotho (1:1000; Cell Signaling Technology), anti-P53 (1:1,000, Cell Signaling Technology), anti-P21 (1:1000, Cell Signaling Technology), anti-IGF-1R (1:1000, Proteintech), anti-MFN2 (1:1000, Cell Signaling Technology), anti-OPA1 (1:1000, Abcam) and anti-p-DRP-1(Ser616) (1:1000, Cell Signaling Technology) antibodies at 4°C overnight. Then, the cells were incubated with horseradish peroxidase-conjugated anti-rabbit (1:5000) and anti-mouse (1:5000; both from Jackson Labs) antibodies for 1 h at room temperature. Immunoreactive signals were detected using enhanced chemiluminescence reagents (EMD Millipore; Merck KGaA). Finally, densitometric analysis of the protein bands was performed using ImageJ (NIH).

Mitochondrial ROS in INS-1 cells was determined by Mito-Sox staining. INS-1 cells were cultured in 24-well plates with different treatments. Then, the cells were incubated with 5 μM Mito-Sox (Invitrogen, M36008) for 20 min at 37°C in the dark. Finally, the sample was randomly photographed, and the fluorescence intensity was analysed from five fields of each group using ImageJ software in three independent experiments.

ATP levels in INS-1 cell lysates were measured using a luminometer (Synergy H4, BioTek Instruments, Inc., United States) according to the manufacturer’s instructions (S0027, Beyotime Biotechnology). Total protein was extracted from INS-1 samples for normalization before ATP assay. According to the manufacturer’s instructions, after centrifugation to remove cell debris, the supernatant was mixed with the substrate solution. The luminescence of the solution was measured using a luminometer with an integration time of 1 s per well. Protein content was determined using the BCA Protein Assay Kit (P0012S, Beyotime), and the ATP concentration was then converted to nanomoles of ATP per microgram of protein.

Cell viability was assessed by CCK8 assay. For the CCK8 assay, experiments were performed in 96-well plates, and INS-1 cells were serum-starved overnight before treatment with various concentrations of Dox for 24 h. Samples of conditioned media were collected for the measurement of viability (CCK8 assay kit, Beyotime), an indicator of cell viability.

Pancreatic sections (4 μM) were incubated with rabbit anti-Klotho (1:100; Cell Signaling Technology), rabbit anti-IGF-1R (1:200; Proteintech), and mouse anti-insulin (1:1000; Proteintech), followed with Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (1:1000; Abcam) and Goat Anti-Mouse IgG H&L (Cy3^®) (1:1,000; Abcam). All immunofluorescent images were captured by an Olympus upright BX50 fluorescence microscope (Olympus, Huashan Hospital, Shanghai, China) at ×40 magnification.

Cells were cultured in 12-well cluster dishes and incubated with 1 μM or 2 μM Klotho in the presence of 30 nM doxorubicin for 16 h. Cells were washed three times with HEPES balanced salt solution (HBSS) containing 125 mM NaCl, 5.9 mM KCl, 1.28 mM CaCl₂, 1.2 mM MgCl₂, 25 mM HEPES (pH 7.4), 2.8 mM glucose and 0.1% fatty acid free BSA (Boehringer Mannheim, GmbH, Germany). The cells were then preincubated with HBSS for 30 min at 37°C and then stimulated with different concentration of glucose (2.8, 16.7 mM) for 2 h at 37°C. Each condition was performed insulin radioimmunoassay. Insulin radioimmunoassay was conducted according to the kit’s instructions (Crystal Chem Inc.).

Data are expressed as the means ± SEMs. P values were determined by two-tailed Student’s t test or one-way ANOVA with Bonferroni posttest for multiple comparisons. P < 0.05 was considered statistically significant.

It has been reported that cellular senescence can be induced in β-cell line by doxorubicin (Dox) treatment (Aguayo-Mazzucato et al., 2019). We conducted a dose response experiment with different doses of doxorubicin (Dox) in INS-1 cell to determine the optimal dose of Dox we use in this study. Our results showed that the number of positive SA-β-gal cells was significantly increased after 24 h of Dox treatment (Figure 1A). Since senescent cells lose the ability to proliferate, we also examined the expression of Ki67, a widely used marker for cell proliferation. As shown in Figure 1B, the expression of Ki67 decreased significantly. We also analysed the expression of crucial pathway P53-P21 related to cellular senescence. QPCR and Western blot analysis showed that the expression of P53 and P21 was significantly increased after Dox treatment (Figures 1C, D). These results demonstrated that Dox could induced senescence in INS-1 cell. And 30 nM Dox did not exhibit toxicity to INS-1 cell (Supplementary Figure S1). Overall, the concentration of Dox (30 nM) was chosen for the remaining experiments in this study.

In order to investigate the role of KL in senescent β-cells, we examined the expression of KL in INS-1 cells with Dox treatment. As shown in Figures 1E, F, IF staining and Western blot showed that the expression of KL was decreased significantly. These results indicated that KL may play an important role in process of β-cell senescence.

To explore the effect of KL on β-cell senescence, cultured INS-1 cells were treated with 30 nM Dox in the presence of 1 µM or 2 µM recombinant human KL protein for 24 h. Then, an SA-β-gal assay was used to detect the number of senescent cells in each group. Very few INS-1 cells were SA-β-gal positive in the non-treatment control group. The number of SA-β-gal-positive cells in the Dox-treated group was significantly increased, while after KL treatment, the number of SA-β-gal-positive cells was much lower than that in the untreated group (Figure 2).

Next, we performed qPCR and Western blotting experiments to confirm the effect of KL on senescent β-cell characterized by senescence markers. qPCR and Western blotting showed that KL significantly reduced the expression of senescent marker IGF-1R, and the expression of pathway P53-P21 and TNF-α compared to the nontreatment (Figures 3A, B). Moreover, IF staining confirmed that KL significantly reduced the numbers of P53- and P21-positive cells (Figures 3C, D). One of the well-known effects of Dox is its ability to induce DNA damage, which serves as a marker for senescent cells. We examined the DNA damage marker γ-H2AX, and the results showed that Dox indeed promoted the expression of the DNA damage marker γ-H2AX, while 2 μM KL could significantly reduce the expression of γ-H2AX (Figure 3E). All of the above proved that Dox promoted the senescence of INS-1 cells in vitro and KL could reduce senescence.

To explore the underlying mechanism by which KL reduced β-cell senescence, mitochondrial dynamics and ROS generation were initially evaluated (Sahu et al., 2018; Lee et al., 2021). As shown in Figures 4A, B, Western blot analysis and IF staining showed that compared with the control, the expression of the mitochondrial fusion protein MFN2 was upregulated after Dox treatment for 24 h, suggesting that the occurrence of mitochondria fusion and KL significantly reduced this upregulation. At the same time, we assessed changes of mitochondrial fission. The results showed KL treatment upregulated the expression of the mitochondrial fission proteins OPA1 and p-DRP-1 (ser 616), when DRP1 is phosphorylated at Ser616, its GTPase activity increases, promoting mitochondrial fission.

We used Mito-Sox to detect ROS production in INS-1 cells. As shown in Figure 4C, the ROS level was significantly enhanced in cells treated with Dox, suggesting that ROS may be involved in the regulation of INS-1 cell senescence. After KL treatment, ROS production was reduced significantly. Additionally, we used flow cytometry to detect ROS production. As shown in Figure 4D, ROS production was significantly increased in cells treated with Dox, and KL reduced ROS production. We also detected the ATP levels in senescent cells. Our date showed that Dox could reduce ATP content significantly, while KL elevated ATP content in cells treated with Dox (Figure 4E).

We further explored whether KL could regulate insulin secretion in cultured β-cells, we performed GSIS in INS-1 cells treated with Dox and KL. We confirmed that Dox treatment reduced insulin secretion significantly, while insulin secretion increased after KL treatment (Figure 5). The results demonstrated that KL promoted insulin secretion.