Effects of 5-Azacitidine on Dnmt2/Trdmt1 expression levels and endoplasmic reticulum stress in cellular models of insulinoma

Diabetes mellitus affects people all over the world of all ages or social groups making it a worldwide healthcare challenge. Moreover, untreated or badly treated diabetes carries a risk of serious complications including premature death (IDF Diabetes Atlas 9th edition, 2019). The main symptom of diabetes is insulin secretion and/or action disorder leading to hyperglycemia what results in impaired carbohydrate, fat, and protein metabolism (“Diagnosis and Classification of Diabetes Mellitus,” 2013). Dnmt2/ Trdmt1 in its structure and sequence is similar to DNA methyltransferases, however, it has been shown that mainly methylates aspartic acid transfer RNA, specifically at the cytosine-38 residue (Goll et al., 2006; Okano et al., 1998). tRNA methylation ensures accurate protein synthesis, tRNA structure stabilization, codon-anticodon interaction strengthening, and prevention from any mistakes in a frameshift (Hori, 2014). Different tRNA deficiency and various enzymes modifying ribonucleotides in tRNAs can disturb ER function. In the presented study we wanted to explore the potential role of 5-Azacitidine and Dnmt2/Trdmt1 in pancreatic β-cells. To investigate Trdmt1, ER stress, senescence, and aging and potential implications in diabetes insulin production and but also possible promotion of senescent cells elimination, insulinoma pancreatic β-cells were treated with two compounds: hydrogen peroxide, a wellknown pro-oxidant inducing aging, and SISP, and 5-Azacitidine, an inhibitor of DNA methylation and tRNA methylation suppressor at C38 position which is a major Trdmt1 target (Lewinska et al., 2018; Schaefer et al., 2009). In the presented study two insulin secreting pancreatic β-cell lines were treated with hydrogen peroxide and 5-Azacitidine. NIT-1 is an insulinoma cell line established from NOD/Lt (nonobese diabetic) mice and transgenic for the SV40 T-antigen. BetaTC6 is derived from insulinoma transgenic mice expressing SV40 T-antigen. Both cell lines secrete insulin in a response to the presence of glucose in the medium. Moreover, NIT-1 spontaneously develops type 1 diabetes what makes it a good model to understand T1D pathogenesis and treatment (Pearson et al., 2016; Poitout et al., 1995). Presented results indicate 5-AzaC mediated endoplasmic reticulum stress in pancreatic βcells thus UPR and UPS activation. 5-AzaC induced tRNA methylation inhibition at the Trdmt1 target site in T1D representative leads to ER stress mediated autophagy pathway which prevents apoptosis and maintains proliferation rate. Furthermore, 5-AzaC boosted 5 insulin production in the non-obese diabetic cell line. It has been shown that BetaTC6 cells are more vulnerable to stress and HP induced excessive NO production, ER stress, and related activation of apoptotic cell death pathway and decreased proliferation. Moreover, HP mediated premature senescence, signaling inhibition, and proliferation blockage of senescent cells indicates its senostatic activity and HP/5-AzaC mediated selective elimination of senescent cells treatment demonstrates senolytic activity. Additionally, Trdmt1 may help to cope with the cellular stress induction either by augmented translocation to the cytoplasm or increased biosynthesis. 5-AzaC modulates insulin secretion by senescent insulinoma cells and enhanced insulin production in the NOD cell line may be considered as a factor preventing T1D. The combined therapy of prooxidants and 5-AzaC can be studied as a promising candidate for insulinoma treatment.