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Transcriptome sequencing for exploring the mechanism of islet cell protection by anthocyanin.

ABSTRACT: We applied GSIS, immunofluorescence staining, WB, and flow cytometry to observe the protective effects of C3G on palmitate-induced beta cell apoptosis and insulin deficiency, after which we explored the mechanism using RNA sequencing (RNA-seq) and quantitative real-time PCR (qPCR), and finally validated the mechanism using Western Blot (WB) and small interfering RNA (siRNA).The results of GSIS on both INS-1E cells and mouse primary islets plus a series of experiments related to cell apoptosis showed that C3G could restore the damage caused by lipotoxicity through promoting insulin secretion and alleviating cell apoptosis. RNA-seq and qPCR results indicated that the protective effect of C3G was most likely mediated by repressing ER stress pathways, especially the PERK pathway, while partly due to elevated expression of insulin secretion-related genes. Finally, the results of knockdown of the pro-apoptotic marker CHOP in PERK pathway further confirmed the above findings.Conclusion: C3G may act though reducing ER stress to exert its protective impact on pancreatic beta cells under lipotoxicity. This research provides favorable evidence for the therapeutic application of C3G in T2DM.

ORGANISM(S): Rattus norvegicus

PROVIDER: GSE196704 | GEO | 2022/05/18

REPOSITORIES: GEO

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Project description:Protein phosphatase 2A (PP2A) is one of the most common serine/threonine phosphatases in mammalian cells and primarily functions to regulate cell signaling, glycolipid metabolism, and apoptosis. Its PP2A catalytic subunit (PP2Ac) plays an important role in its function. Nonetheless, at present, there are only a few reports on the regulatory role of PP2Ac in pancreatic β-cells under lipotoxicity. Mouse pancreatic insulinoma (MIN6) cells were transfected by lentiviruses to generate PP2Ac knockdown cells and incubated with palmitate (PA) to establish the lipotoxicity model. Serine/Threonine Phosphatase Assay System kit, Cell Counting Kit-8 (CCK-8), flow cytometry, enzyme-linked immunosorbent assay (ELISA), Western Blotting (WB) and other techniques were used to measure PP2A activity, cell viability, apoptosis, oxidative stress, and insulin secretion. An animal model of lipotoxicity with knockdown of PP2Ac was established by a high-fat diet (HFD) after using adeno-associated viruses (AAV) to interfere with PP2Ac expression in mouse pancreatic tissues. Serine/Threonine Phosphatase Assay System kit, ELISA, pancreatic tissue immunofluorescence and other techniques were used to measure PP2A activity in pancreatic tissue, serum insulin level and the proliferation of mouse pancreatic β-cells. We found that PP2Ac knockdown inhibited lipotoxicity-induced PP2A hyperactivation, increased the resistance of pancreatic β-cells to lipotoxicity, decreased PA-induced apoptosis in MIN6 cells, protected the function of both the endoplasmic reticulum (ER) and mitochondria, and improved insulin secretion. By mRNA sequencing and Western blotting analysis, we hypothesized that the protective effects of PP2Ac knockdown in MIN6 cells may be mediated by the MAPK pathway. Moreover, the results obtained from animal experiments suggest that the specific knockdown of pancreatic PP2Ac could effectively attenuate HFD-induced insulin resistance and reduce the compensatory proliferation of pancreatic β-cells in mice. The present study revealed the effects and mechanisms of interfering with PP2Ac gene expression on pancreatic β-cells in vivo and in vitro, which might provide insights for the treatment of type 2 diabetes in the clinic.

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Transcriptome sequencing for exploring the mechanism of islet cell protection by anthocyanin.

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