LncRNA-Malat1 is involved in lipotoxicity-induced β-cell dysfunction and the therapeutic effect of exendin-4 via Ptbp1

Increasing evidence indicates that long noncoding RNAs (lncRNAs) have crucial roles in various biological processes. However, the contribution of lncRNAs to β-cell dysfunction and their roles in diabetes therapeutics remain poorly understood. The aim of this study was to identify the lncRNAs dysregulated in diabetic islets and to explore the lncRNAs involved in β-cell function as potential therapeutic targets. By using RNA-sequencing and real-time PCR, we identified thousands of lncRNAs in the islets of db/db mice and db/m littermate mice. Among the differentially expressed lncRNAs, lncRNA-Malat1 (metastasis associated lung adenocarcinoma transcript 1) was reduced in the islets of db/db mice and palmitate-treated MIN6 cells. The results of TUNEL, western blot and flow cytometric analyses and GSIS assays revealed that Malat1 knockdown significantly induced β-cell apoptosis and inhibited insulin secretion. Mechanistically, RNA-immunoprecipitation showed that Malat1 enhanced Ptbp1 (polypyrimidine tract-binding protein 1) protein stability by direct interaction, thereby adjusting the ratio of PKM (pyruvate kinase muscle) isoforms 1 and 2 (PKM1/PKM2). Moreover, luciferase assay and chromatin immunoprecipitation indicated that Malat1 was transcriptionally activated by Pdx1, through which exendin-4 alleviated lipotoxicity-induced β-cell damage. In summary, our findings suggested the involvement of Malat1 in β-cell dysfunction under diabetic conditions via the Malat1/Ptbp1/PKM2 pathway. In addition, exendin-4 ameliorated β-cell impairment by Pdx1-mediated Malat1 upregulation. Hence, Malat1 may serve as a therapeutic target for the treatment of type 2 diabetes.Funding provided by: the Department of Finance of Guangdong ProvinceCrossref Funder Registry ID: Award Number:Six-week-old male C57BL/KsJ db/db mice (n=40) and their age-matched db/m littermate controls (n=20) were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China). Random blood glucose levels were monitored once a week. At 8 weeks old, C57BL/KsJ db/db mice were randomly subdivided to receive either exendin-4 (0.1 mg/kg/d; Sigma-Aldrich, St. Louis, MO) or PBS, to serve as controls, by intraperitoneal injection during the light cycle for 2 weeks. Glucose tolerance tests (GTTs) and insulin tolerance tests (ITTs) were performed after 2-week intervention. For the IPGTTs, mice were fasted overnight and injected intraperitoneally with glucose at a dose of 2 g/kg body weight. For the IPITTs, the animals were intraperitoneally administered with insulin (1U/kg body weight, Novolin R, Novo Nordisk INC, Denmark) after 6-h fasting. Blood samples were collected from the tail vein at 0, 30, 60, 90 and 120 min after the injection of glucose or insulin, and the blood glucose levels were measured with an ACCU-CHEK Performa glucometer (Roche Diabetes Care GmbH, Germany). At the end of intervention, the mice were sacrificed under anesthesia after fasting overnight, blood samples were collected for serum collection, and the islets were isolated. The level of serum insulin was measured by a mouse insulin ELISA kit (Mercodia, catalog no. 10-1247-01) (17) according to manufacturer's protocols. Triglyceride, low-density lipoprotein cholesterol and total cholesterol contents in the serum were quantified with a triglyceride GPO-POD assay kit, low-density lipoprotein cholesterol assay kit and total cholesterol assay kit, respectively, with a Beckman Coulter biochemical analysis system. Pancreas specimens were excised and fixed with 4% paraformaldehyde for histological evaluation. Ten-week-old female C57BL/6 mice (n=20) were also purchased from the Model Animal Research Center of Nanjing University for islet isolation. All animal care and experimental procedures were approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University