Identification of key differentially expressed proteins in the pancreas of obese T2DM mice

We first established a mouse model of obese T2DM through high-fat feeding [28]. Compared with the Ctrl group, the Ob-DM group presented greater body weights (Fig. 1A). Correspondingly, the IPGTT and IPITT curves, along with their areas, indicated abnormal glucose tolerance and reduced insulin sensitivity in the Ob-DM group (Fig. 1B-C, S1A-B). Moreover, compared with the Ctrl group, the Ob-DM group presented increased HOMA-IR and decreased HOMA-β indices (Fig. S1C, D), increased fasting insulin levels and increased plasma TG and FFA levels (Fig. S1E-G). These results indicate that Ob-DM group mice display significant physical and metabolic characteristics of obesity and T2DM. Double immunofluorescence staining of insulin and glucagon in pancreatic tissue revealed that the insulin fluorescence intensity per islet area was significantly weaker in the Ob-DM group than in the Ctrl group (Fig. S1H-I). However, the glucagon fluorescence intensity per islet area was stronger in the Ob-DM group (Fig. S1H, J). The islet area in the Ob-DM group was significantly larger in the pancreatic section (Fig. S1K). These results suggesting that islet area showed an increase, β-cells exhibited dysfunction, α-cells enhanced the secretion of glucagon in obese T2DM mice.

To investigate the underlying mechanisms of impaired pancreatic β-cell function in obese T2DM, the distal portion of the pancreas was harvested from Ctrl and Ob-DM group mice for proteomic sequencing analysis. This proteomic analysis revealed the differential expression of 494 proteins, with upregulation noted in 383 instances and downregulation in 110 instances. The corresponding heatmaps and volcano plots delineating these protein expression patterns are depicted (Fig. 1D, E). According to the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, ferroptosis was the most significantly enriched pathway in the proteome (Fig. 1F). We obtained and analyzed two transcriptome datasets from normal and obese T2DM mouse pancreas tissues from the NCBI GEO database (GSE153222 and GSE122984). Venn diagram analyses were employed to identify commonalities in the upregulated genes (or proteins) across the three datasets, yielding two potential candidate genes, NFAT5 and SNCA, for further study (Fig. S1L). The SNCA is expressed primarily in neuronal cells, where it plays a role in synaptic activity [31]. Extensive literature indicates that NFAT5 is closely associated with diabetes, as it is highly expressed in various diabetic complications and contributes to their progression [19,20,21]. The expression patterns of NFAT5 within the proteomic sequencing data are illustrated in a violin plot (Fig. S1M). A detailed examination of the correlation between NFAT5 expression and the expression of key ferroptosis proteins (ACSL4, GPX4, and FTH1) was performed. The analysis revealed a positive correlation between NFAT5 and the proferroptosis protein ACSL4 and a negative correlation between NFAT5 and the antiferroptosis proteins GPX4 and FTH1 (Fig. 1G-I). These findings collectively underscore the close relationship between NFAT5 and ferroptosis, which may be involved in the pathogenesis of pancreatic damage in obese T2DM. Therefore, NFAT5 was selected for further research on the mechanisms underlying β-cell damage in obese T2DM.

Fig. 1

NFAT5 and ferroptosis may be related to obese T2DM-related pancreatic β-cell damage. (A) Monitoring of body weight per mouse throughout the experiment. (B, C) IPGTTs and IPITTs were performed on Ctrl group mice and Ob-DM group mice for 24 weeks. (D) Heatmap depicting the top 50 differentially expressed proteins. (E) Volcano plot illustrating differential protein expression. (F) KEGG pathway enrichment analysis results are represented in a bubble diagram. (G-I) Pearson correlation analysis examining the relationship between NFAT5 expression and the expression of ACSL4, GPX4, and FTH1 on the basis of proteomic sequencing data. All the results are presented as the mean ± standard deviation (SD). Significance levels are denoted as follows: *p < 0.05, **p < 0.01, ***p < 0.001

Upregulation of NFAT5 expression and ferroptosis in pancreatic β-cells from obese T2DM mice

Compared with the Ctrl group, the Ob-DM group presented significant increases in the expression levels of NFAT5 mRNA and protein in pancreatic tissue, as determined via qPCR and WB analyses (Fig. 2A, B). Double immunofluorescence staining of insulin and NFAT5 in pancreatic tissue revealed that the fluorescence intensity of NFAT5 was notably greater in the islet β-cell areas of the Ob-DM group than in those of the control group (Fig. 2C, D). These results indicate that NFAT5 may primarily participate in the process of β-cell damage in obese T2DM.

At the molecular level, ferroptosis is distinguished by the intracellular accumulation of Fe²⁺, ROS, and lipid peroxidation, concomitant with a reduction in GSH levels. To investigate whether pancreatic β-cells in obese T2DM are subject to ferroptosis, we assessed the concentrations of Fe2+, MDA, and GSH in pancreatic tissue. Our findings indicated that, relative to those in the Ctrl group, the levels of Fe2+ and MDA were elevated in the Ob-DM group, concurrently with a reduction in GSH (Fig. 2E-G). WB analysis of the levels of proferroptosis proteins (ACSL4 and NCOA4) and antiferroptosis proteins (GPX4, SLC7A11, and FTH1) in pancreatic tissues revealed increased ferroptosis in the pancreatic β-cells of the Ob-DM group, as indicated by increased ACSL4 and NCOA4 levels and decreased GPX4, SLC7A11, and FTH1 levels in the Ob-DM group compared with those in the Ctrl group (Fig. 2H-I). We further localized pancreatic β-cells via insulin immunofluorescence and performed DHE (for measuring ROS) or BODIPY C11 (for measuring lipid peroxides) staining. Compared with those in the Ctrl group, notable increases in the ROS (Fig. 2J, K) and lipid peroxide (Fig. 2L, M) levels were observed in the β-cells of the Ob-DM group. TEM was used to examine the morphological features of β-cells, and we observed that β-cells from the Ob-DM group presented pronounced swelling, disrupted mitochondrial cristae, and vacuolar degeneration (Fig. 2N). The combined findings suggest a notable increase in ferroptosis within the β-cells of HFD-fed mice, underscoring the importance of ferroptosis in the development of T2DM. Additionally, we analyzed the number of mature secretory granules in the TEM images of β-cells as previously described [29]. The results showed that compared to the Ctrl group, the number of mature secretory granules within β-cells was significantly decreased in the Ob-DM group (Fig. S1N), indicating a defect in the maturation of insulin granules in β-cells of obese T2DM mice.

Fig. 2

NFAT5 and ferroptosis in β-cells from obese T2DM. (A-B) NFAT5 expression in the pancreas was evaluated via qPCR and Western blot analysis. (C) Insulin (green) and NFAT5 (red) were stained via double immunofluorescence, and the cell nuclei (blue) in the pancreatic tissue were stained blue with DAPI. (D) Quantification of NFAT5 levels within islets normalized to the unit area of the islet. (E-G) Levels of Fe2+, MDA, and GSH in pancreatic tissue. (H) WB was performed to analyze the protein levels of ACSL4, NCOA4, SLC7A11, GPX4, and FTH1 in pancreatic tissue. (I) Quantitative analysis of WB gray values. (J) Insulin immunofluorescence (green) and adjacent slices stained with DHE (red) in pancreatic tissue. (K) Measurement of ROS levels in islets standardized to the islet area. (L) Insulin immunofluorescence (red) and BODIPY C11 staining of adjacent slices (green). DAPI was used to stain the cell nuclei (blue) in the pancreatic tissue. (M) Quantification of lipid peroxide levels within islets normalized to the unit area of the islet. (N) Transmission electron microscopy (TEM) image showing β-cells. All the results are presented as the mean ± SD. Significance levels are denoted as follows: *p < 0.05, **p < 0.01, ***p < 0.001. (C, J, L) Scale bars are 100 μm. (N) From left to right: 5 μm, and 2 μm

PG induced Min6 cell dysfunction via increased ferroptosis

To simulate the microenvironment of T2DM, we established an in vitro model of pancreatic β-cell damage by adding exogenous palmitate and high glucose (PG). We found that the viability of Min6 cells gradually decreased with increasing concentrations and durations of PG treatment (Fig. S2A). PG at a concentration of 200 µM/25 mM for 48 h was selected for subsequent experiments. GSIS was performed to estimate the effect of low concerntration (25/10 µM/mM) PG on the secretion ability of MIN6 cells. The results showed that PG at concentrations of 25/10 µM/mM is capable of inhibiting insulin secretion in MIN6 cells; however, its inhibitory efficacy is weaker compared to that observed with a concentration of 200/25 µM/mM PG. This indicates that the impairment of insulin secretion function in MIN6 cells, as induced by PG, is not solely attributable to its impact on cell viability.

We employed Fer-1 and DFO, two classic inhibitors of ferroptosis, along with the ferroptosis inducer erastin to explore the impact of ferroptosis on MIN6 cells subjected to PG treatment. Exposure to PG and erastin decreased cell viability, increased the levels of MDA and Fe2+ and decreased the levels of GSH in MIN6 cells. In contrast, Fer-1 and DFO improved cell viability, rescued the increased levels of MDA and Fe2+, and rescued the decreased levels of GSH in PG-treated MIN6 cells (Fig. 3A-D). In the GSIS assay, Fer-1 and DFO reversed the inhibition of insulin secretion induced by PG, whereas Erastin exacerbated the impairment of insulin function in Min6 cells (Fig. 3E). PG and Erastin treatment significantly increased the levels of ROS and lipid peroxides, as evidenced by fluorescence staining and flow cytometry analysis of MIN6 cells. Fer-1 or DFO reversed the increase in ROS and lipid peroxides in MIN6 cells treated with PG (Fig. 3F-I).

Moreover, key proteins involved in ferroptosis were detected to further assess the underlying molecular mechanism in PG-treated MIN6 cells. After treatment with PG, the levels of the proferroptosis proteins ACSL4 and NCOA4 significantly increased, and the levels of the antiferroptosis proteins FTH1, GPX4, and SCL7A11 significantly decreased; these changes were the same as those observed after treatment with erastin. Fer-1 and DFO reversed the increase in ACSL4 and NCOA4 and the decrease in FTH1, GPX4, and SCL7A11 after MIN6 cells were treated with PG (Fig. 3J, K). These results suggested that PG induced ferroptosis in MIN6 cells. These results further revealed that ferroptosis is a key mechanism involved in PG-induced damage to MIN6 cells.

Fig. 3

Ferroptosis is involved in the PG-induced functional impairment of MIN6 cells. Min6 cells were exposed to 200 µM/25 mM PG for 48 h after being pretreated with 5 µM Fer-1, 10 µM DFO, or 5 µM erastin for 6 h, after which cell viability was measured via a CCK8 assay (A). DMSO served as a solvent control. The levels of MDA (B), Fe2+ (C), and GSH (D) were measured in Min6 cells treated with PG and pretreated with Fer-1, DFO, or erastin. To estimate the secretion ability of MIN6 cells under different treatment, GSIS was performed with or without 25mM glucose stimulation (E). Intracellular ROS can be detected through DCFHDA staining, which involves fluorescence imaging (F), and flow cytometry (G). Assessment of lipid peroxides through BODIPY C11 fluorescence imaging (H) and flow cytometry analysis (I). WB analysis was performed to assess the protein expression of ACSL4, NCOA4, GPX4, SLC7A11, and FTH1 in Min6 cells under various conditions (J), and band intensities were quantified with ImageJ (K). All the results are presented as the mean ± SD. Significance levels are denoted as follows: *p < 0.05, **p < 0.01, ***p < 0.001. (F) Scale bars are 30 μm and (H) 50 μm

NFAT5 is involved in PG-induced Min6 cell ferroptosis

NFAT5, a regulatory protein, is translocated into the nucleus for transcriptional activity [30, 31]. NFAT5 translocation into the nucleus was examined in MIN6 cells treated with PG by exposing the cells to PG for different durations (12 h, 24 h, 48 h, and 72 h). Proteins from the cytoplasm and nucleus were separated with a kit designed for extracting nuclear and cytoplasmic components, and the levels of NFAT5 in both the cytoplasm and nucleus compartments were measured via WB. The findings revealed a continuous increase in the levels of the NFAT5 protein in the nucleus and total in Min6 cells treated with PG. Notably, cytoplasmic NFAT5 protein levels were elevated at 12 h and 24 h but decreased after 48 h and 72 h of PG treatment (Fig. 4A, B). Similarly, the total level of NFAT5 mRNA in PG-treated MIN6 cells initially increased for up to 24 h but then gradually decreased (Fig. 4C).

Immunofluorescence staining was performed to further confirm the subcellular localization of NFAT5 after treatment with PG at different time points. Initially, NFAT5 was predominantly observed in the cytoplasm, but after 24 h of PG treatment, it gradually translocated to the nucleus. Additionally, the total NFAT5 fluorescence intensity in the cells increased slightly (Fig. 4D). These findings indicated that NFAT5 was significantly upregulated and translocated into the nucleus upon treatment of Min6 cells with PG. To investigate whether the expression of NFAT5 is osmotically dependent, we treated Min6 cells with mannitol equilibrated with PG. Compared with the control group, the mannitol group did not affect the viability or expression of NFAT5 in Min6 cells (Fig. S3A-C).

Preliminary experiments validated the knockdown efficiency of the three shNFAT5 plasmids in Min6 cells (Fig. S3D-F). shNFAT5-2 was selected as the vector for use in subsequent experiments. NFAT5 knockdown effectively rescued the reduction in insulin secretion induced by PG treatment (Fig. 4E). Moreover, shNFAT5 reversed the decreases in cell viability and GSH levels and the increases in MDA and Fe2+ levels in MIN6 cells treated with PG (Fig. 4F-I). NFAT5 knockdown also reduced the elevated levels of ROS (Fig. 4J-L) and lipid peroxides (Fig. 4M-O) in Min6 cells treated with PG. These results indicated that knockdown of NFAT5 can alleviate ferroptosis and improve the reduction in insulin secretion induced by PG in pancreatic β-cells.

WB analysis revealed that inhibition of NFAT5 reversed the PG-induced upregulation of ACSL4 and downregulation of GPX4 (Fig. 4P, Q). However, the levels of NCOA4, FTH1, and SLC7A11 were not affected by NFAT5 knockdown in Min6 cells treated with PG (Fig. S3G, H). These results corroborate the involvement of NFAT5 in PG-induced ferroptosis through the regulation of ACSL4 and GPX4 rather than NCOA4, FTH1, or SLC7A11.

Fig. 4

NFAT5 is a key regulator of ferroptosis in PG-treated cells. NFAT5 expression was analyzed in Min6 cells exposed to 200 mM/25 mM PG for 12, 24, 48, or 72 h. (A) Nuclear and cytoplasmic proteins were isolated and subjected to WB, with β-actin and lamin B1 serving as loading controls for the cytoplasmic and nuclear fractions, respectively. (B) Detection of total NFAT5 protein expression by WB, with β-actin serving as a loading control. (C) Quantification of NFAT5 mRNA expression. (D) Immunofluorescence was used to observe the presence and levels of NFAT5 in Min6 cells exposed to 200 mM/25 mM PG for 12, 24, 48, or 72 h. (E) Insulin secretion was evaluated through GSIS tests in MIN6 cells exposed to PG after transfection with shNC or shNFAT5 plasmids. (F) Detection of cell viability. (G-I) The following evaluations involved measuring the concentrations of MDA, GSH, and Fe2+. (J-L) ROS levels were evaluated via DCFHDA staining through fluorescence imaging (J), measurement of ROS levels (K), or flow cytometry analysis (L). Lipid peroxides were detected via BODIPY C11 staining, fluorescence imaging (M), measurement of lipid peroxide levels (N), or flow cytometry analysis (O). The protein levels of NFAT5, ACSL4, and GPX4 were measured via WB, and the band intensities were quantified via ImageJ (P, Q). All the results are presented as the mean ± SD. Significance levels are denoted as follows: ns = nonsignificant difference, *p < 0.05, **p < 0.01, ***p < 0.001. (D, J) Scale bars are 30 μm and (M) 50 μm, respectively

NFAT5 regulates the transcription of PRDX2 in MIN6 cells by targeting the promoter region of PRDX2

To elucidate the involvement of NFAT5 in the regulation of ferroptosis, we utilized hTFtarget (http://bioinfo.life.hust.edu.cn/hTFtarget#!/) to predict the target genes of NFAT5. Venn diagrams revealed 14 intersecting genes by intersecting the potential target genes of NFAT5 with differentially expressed proteins (DEPs) (Fig. 5A). Previous research has shown that PRDX2 attenuates mitochondria-associated ferroptosis by suppressing oxidative stress, iron overload, and lipid peroxidation. Analysis of protein interactions in the STRING database (http//string-db.org) revealed a strong correlation between PRDX2 and critical ferroptosis-related proteins (Fig. S4A).

Double-immunofluorescence staining was performed to assess PRDX2 expression in β-cells. Compared with that in the Ctrl group, the expression of PRDX2 in the β-cell area was decreased in the Ob-DM group (Fig. 5B, C). Consistent with this observation, WB and proteomic sequencing analysis confirmed the decreased expression of PRDX2 in the Ob-DM group (Fig. 5D and S4B). qPCR and WB analyses revealed that the mRNA and protein levels of PRDX2 significantly increased after NFAT5 knockdown in Min6 cells treated with PG (Fig. 5E, F). To elucidate the role of NFAT5 in the transcriptional regulation of PRDX2, we examined the PRDX2 promoter region in the UCSC Genome database and identified a specific segment from − 2000 to + 100 base pairs around the transcription start site (TSS). Afterward, we constructed a plasmid that included the − 2000 to + 100 segment connected to a luciferase reporter vector, named WT-PRDX2P-Luc, which was designated the PRDX2 promoter luciferase reporter gene. Through a dual-luciferase reporter assay, we observed increased PRDX2 promoter activity upon NFAT5 knockdown in PG-treated MIN6 cells (Fig. 5G). Inadditional, an NFAT5 overexpression plasmid was constructed and its successful verification was achieved in MIN6 cells. Dual-luciferase reporter assay found that the overexpression of NFAT5 can suppress the activity of the PRDX2 promoter (Fig. S4C, D). These results suggesting that PRDX2 promoter activity is inhibited by NFAT5.

To further delineate NFAT5 binding sites within the PRDX2 promoter, we utilized the JASPAR database for prediction (Fig. 5H). We focused on the − 2000 to + 100 bp upstream regions of the PRDX2 transcription start site and identified putative binding sites (BS 1–3) (Fig. 5I). Afterward, we created three pGL3 luciferase reporter constructs with mutated BS 1–3 sequences (Fig. 5J). In PG-treated MIN6 cells, the luciferase reporter assay revealed that the luciferase activity of the BS1-mut-PRDX2P-Luc group was significantly greater than that of the other groups (Fig. 5K). To further confirm that NFAT5 binds to these sites, we performed ChIP assays targeting BS1, BS2, and BS3. Agarose gel electrophoresis confirmed the appropriate size range of the sheared DNA fragments (200–2000 bp) (Fig. 5L). Subsequent ChIP‒PCR analyses demonstrated the specific interaction of NFAT5 with the BS1 region (-1725–1736 bp) of the PRDX2 promoter (Fig. 5M, N). These findings underscore the direct interaction of NFAT5 with the BS1 region of the PRDX2 promoter, indicating that NFAT5 mediates the transcriptional inhibition of PRDX2 in MIN6 cells.

Fig. 5

NFAT5-mediated transcriptional regulation of PRDX2. (A) NFAT5 target genes can be predicted via a Venn diagram, which involves integrating our proteomic data with predicted targets from http://bioinfo.life.hust.edu.cn/hTFtarget#!/. Intersection analysis revealed 14 genes of interest. (B) Insulin and PRDX2 were detected via dual immunofluorescence staining, and DAPI was used to counterstain the nuclei. (C) Quantitative assessment of islet PRDX2 levels normalized to islet area (fluorescence intensity per unit of islet area). (D) WB was performed to analyze the protein expression of PRDX2. (E, F) The levels of PRDX2 mRNA and protein were assessed via qPCR and WB analysis after PG-treated MIN6 cells were transfected with shNC or shNFAT5 plasmids. (G) Luciferase reporter assays were performed to assess PRDX2 promoter activity after transfecting PG-treated MIN6 cells with shNC or shNFAT5 plasmids. (H) The NFAT5 binding motif sequence logo was retrieved from the JASPAR database. (I) JASPAR predicted three possible NFAT5 binding sites in the PRDX2 promoter region. (J) Mutations were introduced into the predicted NFAT5 binding sites to generate mutant promoters. (K) Luciferase reporter assays were conducted to assess the functionality of altered PRDX2 promoters in MIN6 cells treated with PG. (L) Chromatin immunoprecipitation (ChIP) experiments were conducted in MIN6 cell lysates before and after sonication, followed by agarose gel electrophoresis. (M) Common PCR was conducted after the ChIP experiment, which utilized three sets of primers to explore possible NFAT5 binding sites in the PRDX2 promoter region. (N) Quantitative analysis of the amount of DNA pulled down via qPCR. All the results are presented as the mean ± SD. Significance levels are denoted as follows: *p < 0.05, **p < 0.01, ***p < 0.001. Scale bars are 100 μm

NFAT5 promoted ferroptosis by inhibiting PRDX2 in PG-treated MIN6 cells

To further elucidate the relationship between NFAT5 and PRDX2, we performed a rescue experiment to assess the combined effect of coknockdown of NFAT5 and PRDX2 on ferroptosis in PG-treated MIN6 cells. Preliminary experiments validated the knockdown efficiency of the three shPRDX2 plasmids in Min6 cells (Fig. S4E-G). shPRDX2-3 was selected as the vector for use in subsequent experiments. WB analysis revealed that shNFAT5 significantly increased the expression of PRDX2, whereas shPRDX2 had no obvious effect on NFAT5 expression in PG-treated MIN6 cells. Moreover, PRDX2 knockdown reversed the decrease of GPX4 expression and the increase of ACSL4 expression caused by NFAT5 knockdown in PG-treated MIN6 cells (Fig. 6A, B). CCK8 and GSIS assays revealed that NFAT5 knockdown greatly enhanced cell viability and insulin secretion, but PRDX2 knockdown eliminated the beneficial impact of NFAT5 knockdown in Min6 cells treated with PG (Fig. 6C, D). Subsequent studies revealed that PRDX2 knockdown reversed the decrease in MDA and Fe2+ levels and the increase in GSH levels induced by NFAT5 knockdown in PG-treated MIN6 cells (Fig. 6E-G). Further analysis via DCFHDA and BODIPY C11 staining demonstrated that the decreases in ROS (Fig. 6H-J) and lipid peroxides (Fig. 6K-M) induced by NFAT5 knockdown were reversed upon knockdown of PRDX2 in PG-treated MIN6 cells.

JC-1 was employed to assess mitochondrial membrane potential (MMP), with a reduction in the red/green fluorescence intensity ratio signifying mitochondrial depolarization. The findings indicated that PG treatment resulted in a decrease in MMP in MIN6 cells, aligning with the features of mitochondrial dysfunction during ferroptosis. The presence of shNFAT5 alleviated the reduction of MMP in MIN6 cells subjected to PG treatment. Conversely, shPRDX2 counteracted the effect of shNFAT5 (Fig. S4H, I). Overall, these results indicate that NFAT5 suppresses the expression of PRDX2, contributing significantly to the worsening of ferroptosis in MIN6 cells treated with PG.

Fig. 6

Silencing of PRDX2 eliminates the defensive impact of NFAT5 knockdown in Min6 cells treated with PG. PG treatment was administered to Min6 cells following transfection with shNFAT5 plasmids either individually or in conjunction with shNFAT5 and shPRDX2 plasmids. (A, B) Western blotting was conducted to assess the protein expression of PRDX2, NFAT5, ACSL4, and GPX4, and band intensities were quantified with ImageJ. (C) GSIS assays were conducted to assess insulin secretion in MIN6 cells subjected to different treatments. (D-G) The following evaluations involved measuring cell viability and the concentrations of MDA, GSH, and Fe2+ under various treatment conditions. Intracellular ROS were detected via DCFHDA staining, followed by fluorescence imaging (H), measurement of ROS levels (I), or flow cytometry (J). Lipid peroxide levels were assessed via BODIPY C11 staining (K, L) and flow cytometry (M). All the results are presented as the mean ± SD. Significance levels are denoted as follows: *p < 0.05, **p < 0.01, ***p < 0.001. (H) Scale bars are 30 μm and (K) 50 μm

Suppression of NFAT5 relieves pancreatic β-cell ferroptosis in vivo

We assessed the specificity of AAV8 carrying the RIP2 vector for pancreatic β-cell expression by infecting mice with either AAV8-RIP2-GFP or AAV8-empty. Pancreatic β-cells were identified via insulin immunofluorescence labeling in the pancreatic tissue of the mice. Our results revealed specific GFP expression within pancreatic β-cells following AAV8-RIP2-GFP vector infection, while no GFP expression was observed in liver tissues (Fig. S5A). The ob/ob mouse is a classic obese T2DM mouse model that typically exhibits obesity, hyperlipidemia, hyperglycemia, and impaired glucose tolerance at 6 weeks [32]. To validate the biological role of NFAT5 in pancreatic β-cells in vivo, we constructed AAV8-RIP2-miR30-shNFAT5 to specifically knockdown NFAT5 expression in the pancreatic β-cells of ob/ob mice. Six-week-old ob/ob mice were intraperitoneally injected with either AAV8-RIP2-miR30-shNFAT5 (RIP2-shNFAT5) or AAV8-RIP2-miR30-shNC (RIP2-shNC). After 4 weeks, the mice were collected for subsequent experiments.

We observed no significant difference in body weight between the RIP2-shNC group and the RIP2-shNFAT5 group (Fig. S5B). However, compared with those in the RIP2-shNC group, the insulin levels in the RIP2-shNFAT5 group were greater after 2 h of refeeding (Fig. 7A). The results from the IPGTT and IPITT curves and their areas indicated that the RIP2-shNFAT5 group exhibited superior glucose tolerance and insulin sensitivity compared with the RIP2-shNC group (Fig. 7B, C and Fig. S5C, D). Compared with the RIP2-shNC group, the RIP2-shNFAT5 group presented lower expression of NFAT5 (Fig. 7D), whereas the expression of PRDX2 (Fig. 7E) was greater, and the content of insulin (Fig. 7F) was also increased in the β-cell region. These results suggest that the knockdown of NFAT5 by RIP2-shNFAT5 in β-cells of obese T2DM mice leads to increased expression of PRDX2 and increased insulin content.

WB analysis was used to evaluate the occurrence of ferroptosis in β-cells and revealed a decrease in NFAT5 and ACSL4 expression and an increase in GPX4 and PRDX2 expression in the RIP2-shNFAT5 group (Fig. 7G, H), which was consistent with our in vitro findings. Moreover, measurements of MDA, Fe2+ levels, and GSH levels in pancreatic tissues revealed significant reductions in MDA and Fe2+ levels, accompanied by increased GSH levels in the RIP2-shNFAT5 group (Fig. 7I-K). Staining with DHE or BODIPY C11 revealed decreased ROS and lipid peroxide levels in the β-cells of the RIP2-shNFAT5 group (Fig. 7L, M). To evaluate the β-cell architecture in more detail, TEM was performed. The mitochondrial membranes of β-cells in the RIP2-shNC group exhibited unclear morphology, mitochondrial edema, reduced cristae, and vacuoles in numerous cristae. However, these mitochondrial damage features were ameliorated in the RIP2-shNFAT5 group (Fig. 7N). The number of mature secretory granules also was significantly increased in the RIP2-shNFAT5 group compared to the RIP2-shNC group (Fig. S5E). These observations indicate that pancreatic β-cell-specific NFAT5 knockdown promotes PRDX2 expression and inhibits ferroptosis in pancreatic β-cells from obese T2DM mice. Moreover, we observed improvements in β-cells granules maturity and insulin secretion in obese T2DM mice.

Fig. 7

Effect of specific knockdown of NFAT5 in pancreatic β-cells from ob/ob mice. RIP2-shNC: AAV8-RIP2-miR30-shNC; RIP2-shNFAT5: AAV8-RIP2-miR30-shNFAT5. (A) Intraperitoneal injection of the RIP2-shNC or RIP2-shNFAT5 vector in ob/ob mice. After 4 weeks, insulin levels were measured in fasted and 2-hour refed mice. (B) Blood glucose levels after the IPGTT. (C) Blood glucose levels after the IPITT. (D-F) Insulin was marked green by immunofluorescence staining. NFAT5, PRDX2, or glucagon was marked in red respectively. NFAT5, PRDX2, or glucagon intensity was measured per islet area. (G) The protein levels of NFAT5, PRDX2, GPX4, and ACSL4 in the pancreas of these mice were measured via WB. (H) Quantitative analysis of WB gray values. (I-K) MDA, Fe2+ and GSH levels in pancreatic tissue were measured. (L) Insulin immunofluorescence (green) and adjacent slices stained with DHE (red) in pancreatic tissue. The measurement of ROS levels in islets was standardized to the islet area. (M) Insulin immunofluorescence (red) and BODIPY C11 staining of adjacent slices (green). The lipid peroxide levels within the islets were quantified and normalized to the unit area of the islet. (N) Representative TEM images of pancreatic β-cells. All the results are presented as the mean ± SD. Significance levels are denoted as follows: *p < 0.05, **p < 0.01, ***p < 0.001. (D-F, L, and M) Scale bars are 100 μm. (N) From left to right: 5 μm, and 2 μm, respectively