According to International Diabetes Federation (IDF) reports, there are approximately 536.6 million adult diabetic peoples worldwide and almost half of patients were unaware of their diabetes status (1, 2). Some 90% of diabetic individuals have type 2 diabetes mellitus (T2DM), which is currently the most common type of diabetes mellitus and increases the burden on the state. These patients have higher risks of cardiovascular damage, nerve damage, bacterial infection and other complications (3). The typical features of T2DM are glucotoxicity, lipotoxicity, insulin resistance and islet β cells dysfunction (IBCD) (4). IBCD occurs in progressive stage of T2DM and influences the prognosis of T2DM. Understanding the clear mechanism of IBCD can help us to early identify IBCD and perform targeted therapy in T2DM. At present, studies show that the mechanisms of IBCD in T2DM include mitochondrial dysfunction, inflammation, autophagy, oxidative stress (5). But the specific pathogenesis of IBCD in T2DM is still not completely clear and failure to discerned early.

Ferroptosis, a form of nonapoptotic regulated cell death (RCD), is caused by the lipid peroxide accumulation dependent on iron and antioxidant system dysfunction (6). It is worth noting that islet β cells express low level of antioxidant enzymes such as superoxide dismutase (SOD), glutathione (GSH) peroxidase and catalase, so they are susceptible to suffer from oxidative stress (7). Interestingly, lipid peroxide and iron accumulation were observed in type 2 diabetic islet β cells (8, 9). In addition, the islet β cells significantly died and its capacity of glucose-stimulated insulin secretion reduced, when they were treated with ferroptosis inducer erastin in vitro ( 10). These adverse effects improved in ferroptosis inhibitors Ferrostatin-1- and desferrioxamine-treated islets β cells (10). Thus, IBCD in T2DM probably related to ferroptosis. Probing the underlying molecular mechanism between IBCD in T2DM and ferroptosis may be a breakthrough for its early diagnosis and treatment.

Here, based on the above researches, we screened the key genes by differential expression genes (DEGs) analysis and weighted gene co-expression network analysis (WGCNA) (11) in order to explore the effect of ferroptosis on type 2 diabetic IBCD. These T2DM key genes were intersected with the ferroptosis-related genes (FRGs) to acquire T2DM-FRGs in IBCD. We also validated the T2DM-FRGs using another T2DM islet issues dataset as well as islet single-cell RNA sequencing dataset and predicted microRNAs (miRNAs) regulated T2DM-FRG.

IBCD is considered as the important factor in the progression of T2DM and is also another direction to treat T2DM. Long-term hyperglycemia, hyperlipidemia and uncontrol of blood glucose in patients with T2DM caused the islet β cells damage and even death (4). Some ways of islet β cells death in T2DM, such as apoptosis and autophagy were confirmed in studies (22, 23). But the early diagnosis and treatment of IBCD in T2DM is still a difficult and unsolved problem. As a newly identified iron-dependent form of oxidative cell death, ferroptosis remains unclear in T2DM. The features that type 2 diabetic islet β cells appear low antioxidant activity and iron overload indicated us the possible link between IBCD and ferroptosis (7, 9). We hope to discover the association between the two and detect the target for early diagnosis and treatment in IBCD of T2DM by our study.

MAPK and PI3K-Akt signaling pathways, which have been proved as the pathways regulated ferroptosis, were significantly enriched in KEGG of DEGs in IBCD of T2DM (24, 25). However, no significant ferroptosis-related terms were found in GO of DEGs. We considered that serious ferroptosis may have not occurred in IBCD at pre-middle stage of T2DM, but changes in ferroptosis-related pathways, such as MAPK and PI3K pathways. The differential genes between T2DM and Normal islet tissues were screened by combining DEGs and trait-related genes in WGCNA, which ensured the reliability of data. We obtained 3 T2DM-FRGs (ITGA6, MGST1 and ENO2) from the intersection between differential genes and ferroptosis-related genes. We found MGST1 and ITGA6 is up-regulated, while ENO2 is down-regulated in T2DM. MGST1, not ITGA6 and ENO2, was considered as the key T2DM-FRG after validation in another type 2 diabetic islet issues dataset and single-cell sequencing dataset. This study mays provide reference for research the connection between IBCD and ferroptosis in T2DM.

MGST1 (Microsomal Glutathione S-transferase 1) is a membrane-bound transferase regulates oxidative stress. It mainly located in mitochondria, ER plasma membrane and peroxisome (26). Researchers found that it fighted ferroptosis in pancreatic cancer cells. MGST1 is regulated by Nrf2 (Nuclear Factor erythroid 2-Related Factor 2) and partly combined with ALOX5 (Arachidonate Lipoxygenases 5) to inhibit ferroptosis (27). In addition, MGST1 is up-regulated during ferroptosis (27). We prove that mRNA expression of MGST1 increases in type 2 diabetic IBCD. Thus, it is conceivable to hypothesis that MGST1 mays contribute to alleviate ferroptosis in type 2 diabetic IBCD, which needs to be proved experimentally in the future. ENO2 (Enolase 2 or gamma enolase, also known as neuron-specific enolase, NSE), is a glycolytic enzyme which catalyzes 2-phosphoglycerate to form PEP (Phosphoenolpyruvate). It not only participates in glycolysis and gluconeogenesis, but also is related with inflammation and ferroptosis (28, 29). ENO2 mays influence ferroptosis by neuroglobin (29). A study indicates that ENO2 is positively correlated with insulin secretion and negatively correlated with HbA1c (30). ENO2 is decreased in cultured INS-1 cells treated with high glucose in 12 hours and in type 2 diabetic islet cells (30). These results in vivo and in vitro suggest that ENO2 is a key molecule protect the function of islet β cells. However, mRNA expression of ENO2 did not change in islet single-cell RNA sequencing samples. This may be related to the small single-cell sequencing sample size. Whether ENO2 regulates islet β cells in T2DM through ferroptosis pathway remains to be studied.

In addition, the study about islet issue sequencing has certain limitations. There is no way to know whether the target gene changes are present in β cells because islet issues include a variety of cells, such as α cells, β cells and PP cells. There are many studies about islet single-cell RNA sequencing of T2DM at present (16, 31, 32). Therefore, we used the islet single-cell sequencing dataset to check the target genes again. Single-cell sequencing, though, is more specific to get a lot of β cells and its sequencing data, but contains a few samples in patients for its high cost, which will cause the inability to exclude individual differences. Finally, we selected MGST1 as the potential ferroptosis-related gene in IBCD of T2DM by combing large patient samples of islet issue sequencing datasets and specificity of islet single-cell sequencing dataset.

This study reveals the potential relationship between ferroptosis and IBCD in T2DM based on bioinformatics analysis. It deeply adds to our understanding of type 2 diabetic islet β cells impairment and provides reference for developing targeted therapy for T2DM.

The original contributions presented in the study are included in the article/ Supplementary Material . Further inquiries can be directed to the corresponding authors.

HY designed the study and wrote manuscript, HY and RW conducted the bioinformatic analysis. JW and YW took part in a discussion. XZ and LW amended the manuscript and co-wrote the final draft with HY. All authors contributed to the article and approved the submitted version.

This study was supported by Talent introduction funding project of Guangzhou Overseas Chinese Hospital, Jinan University (no.808026).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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