Inhibition of miR-146a-5p and miR-8114 in Insulin-Secreting Cells Contributes to the Protection of Melatonin against Stearic Acid-Induced Cellular Senescence by Targeting Mafa.

BACKGRUOUND: Chronic exposure to elevated levels of saturated fatty acids results in pancreatic ß-cell senescence. However, targets and effective agents for preventing stearic acid-induced ß-cell senescence are still lacking. Although melatonin administration can protect ß-cells against lipotoxicity through anti-senescence processes, the precise underlying mechanisms still need to be explored. Therefore, we investigated the anti-senescence effect of melatonin on stearic acid-treated mouse ß-cells and elucidated the possible role of microRNAs in this process. METHODS: ß-Cell senescence was identified by measuring the expression of senescence-related genes and senescence-associated ß-galactosidase staining. Gain- and loss-of-function approaches were used to investigate the involvement of microRNAs in stearic acid-evoked ß-cell senescence and dysfunction. Bioinformatics analyses and luciferase reporter activity assays were applied to predict the direct targets of microRNAs. RESULTS: Long-term exposure to a high concentration of stearic acid-induced senescence and upregulated miR-146a-5p and miR- 8114 expression in both mouse islets and ß-TC6 cell lines. Melatonin effectively suppressed this process and reduced the levels of these two miRNAs. A remarkable reversibility of stearic acid-induced ß-cell senescence and dysfunction was observed after silencing miR-146a-5p and miR-8114. Moreover, V-maf musculoaponeurotic fibrosarcoma oncogene homolog A (Mafa) was verified as a direct target of miR-146a-5p and miR-8114. Melatonin also significantly ameliorated senescence and dysfunction in miR-146a-5pand miR-8114-transfected ß-cells. CONCLUSION: These data demonstrate that melatonin protects against stearic acid-induced ß-cell senescence by inhibiting miR-146a- 5p and miR-8114 and upregulating Mafa expression. This not only provides novel targets for preventing stearic acid-induced ß-cell dysfunction, but also points to melatonin as a promising drug to combat type 2 diabetes progression.

Cell-Penetrating Peptide as a Means of Directing the Differentiation of Induced-Pluripotent Stem Cells.

Protein transduction using cell-penetrating peptides (CPPs) is useful for the delivery of large protein molecules, including some transcription factors. This method is safer than gene transfection methods with a viral vector because there is no risk of genomic integration of the exogenous DNA. Recently, this method was reported as a means for the induction of induced pluripotent stem (iPS) cells, directing the differentiation into specific cell types and supporting gene editing/correction. Furthermore, we developed a direct differentiation method to obtain a pancreatic lineage from mouse and human pluripotent stem cells via the protein transduction of three transcription factors, Pdx1, NeuroD, and MafA. Here, we discuss the possibility of using CPPs as a means of directing the differentiation of iPS cells and other stem cell technologies.

Recombinant MafA protein containing its own protein transduction domain stimulates insulin gene expression in IEC-6 cells.

AIMS: MafA, a basic leucine zipper (bZIP) transcription factor, functions as a potent activator of insulin gene transcription in ß-cell. In this paper, we aimed to investigate whether the entire MafA protein has the self-delivery activity, and that the arginine- and lysine-rich sequence in MafA bZIP domain is an efficient protein transduction domain (PTD). MAIN METHODS: Entire MafA protein internalization was evaluated with Western blot and immunofluorescence. The distribution of the PTD-EGFP (enhanced green fluorescence protein) was examined by fluorescent microscope observation. Luciferase reporter assay was used to investigate the effect of the transduced MafA protein on insulin 2 promoter activity. Additionally, we conducted RT-PCR to detect the expression of insulin mRNA in MafA treated IEC-6 cells. KEY FINDINGS: The arginine- and lysine-rich peptide of MafA serves as a novel PTD. PTD-EGFP can permeate into various cell types including Min6 (a ß-cell line), and transduce in a dose- and time-dependent manner. The cellular uptake of MafA PTD can be completely blocked by heparin, whereas cytochalasin D and amiloride were partially effective in blocking the PTD-EGFP protein entry. Transduced intact MafA protein behaves in the same way as the endogenous MafA, stimulating the transcription of insulin promoter and further inducing insulin expression in treated non-ß-cell line (IEC-6). SIGNIFICANCE: These results indicate that the MafA PTD could serve as a therapeutic delivery vehicle, and further suggest that MafA protein transduction could be a valuable strategy for enhancing insulin gene transcription without requiring gene transfer technology.

MafK overexpression in pancreatic beta-cells caused impairment of glucose-stimulated insulin secretion.

MafA is a member of large Maf transcription factors and activates the insulin gene in pancreatic beta-cells. Other large Maf transcription factors, MafB and c-Maf, also express and activate insulin transcription in beta-cells. However, the functional relationship between MafA and other Maf proteins in beta-cells has not been established. In order to suppress the function of large Maf proteins, we generated transgenic mice overexpressing MafK, which act as dominant negative protein in pancreatic beta-cells. These mice showed hyperglycemia at a young age due to impairment of glucose-stimulated insulin secretion. Although the transgenic mice showed an abnormal response in the glucose tolerance test, hyperglycemia was restored in adulthood. Histological analysis revealed islet hypertrophy in adult transgenic mice. Finally, an electrophoretic gel mobility shift assay showed that the DNA-binding activity of endogenous MafA was significantly increased in the MafK transgenic mice. These results indicated that MafA may have relevance to compensatory response.