Abstract
The mechanistic target of rapamycin complex 1 (mTORC1) integrates nutrient availability and hormone/growth factors signaling to regulate cell metabolism; being essential to beta-cell mass and function. The microRNA 199a-3p negatively regulates translation of mTORC1 mRNA and impacts beta-cell function. We investigated how miR-199a expression is regulated in beta-cells by examining expression responses of miR-199a precursors genes (primiR-199-a1, a2 and b) and its mature forms (miR-199a-3p and -5p) and promoter transcriptional activity assays in mouse islets and mouse insulinoma (MIN6) cells treated with different stimuli. We found that islets from male and female mice equally express the mature miR-199a-3p and -5p. However, the primiR expression is different: while primiR-199a1 expression is 2-fold greater than primiR-199a2, primir-199b is barely detected in mouse islets. The same primiR expression profile was found in MIN6 cells except that only the mature miR-199a-3p is expressed. A 2-fold increase in primiR-199a1 and -a2 mRNA levels was observed by 24 h culture of mouse islets in high glucose compared to 5.5 mM. Similar responses to high and low glucose were observed in MIN6 cells as early as 6 h. Interestingly, 30 mM of KCl treatment was sufficient to prevent the low glucose-induced decline in primiR-199-a2 expression but not in 199-a1 in MIN6 cells, indicating that calcium influx was involved. Transcriptional activity studies in MIN6 also reveal that low glucose or 2-DG glucose-induced starvation decreases the primir-199a2 promoter activity and this decrease was rescued by KCl and Tolbutamide (Potassium channel blocker), confirming that calcium influx is playing a role. Indeed, blocking calcium channels with 20 uM Nifedipine reduced primiR-199-a2 promoter activity in MIN6 cells under 25 mM glucose.
In conclusion, we uncover that glucose regulates microRNA 199a family expression in beta cells and that glucose metabolism and calcium influx are involved.
Disclosure
J. Werneck de Castro: None. M. Blandino-Rosano: None. E. Bernal-Mizrachi: None.
