Circulating microRNA-375 as biomarker of pancreatic beta cell death and protection of beta cell mass by cytoprotective compounds

Abstract

Objective: Previous studies demonstrated that circulating microRNA-375 (miR-375) is a suitable plasma biomarker for real-time detection of beta cell death. The present study evaluated the use of this biomarker to assess the beta cytoprotective effect of phenylpropenoic acid glucoside (PPAG), which was previously demonstrated to protect beta cells against various types of injury, and of exendin-4, which is an established antidiabetic drug.

Methods: PPAG or exendin-4 were administered in mice treated with streptozotocin (STZ) to acutely induce beta cell death. Beta cell mass and apoptotic death were measured in pancreatic tissue sections. Circulating miR-375 was measured in blood plasma by RT-qPCR. The release of miR-375 was also measured in vitro by MIN-6 beta cells.

Results: Administration of STZ resulted in measurable circulating levels of miR-375, a decrease in beta cell mass and increase in frequency of apoptotic beta cells. In vitro, there was a good correlation between miR-375 release and the extent of beta cell death. Treatment of mice with PPAG or exendin-4 significantly attenuated STZ-induced loss of beta cell mass and beta cell apoptosis, and normalized the blood level of miR-375.

Conclusions: These findings show the potential use of serological miR-375 measurements to evaluate the beta cytoprotective effect of (potential) antidiabetic drugs in vivo.

Fig 1. Blood glycemia and miR-375 blood levels after STZ injection.

Blood glucose levels were measured at several time points after STZ injection (A). For time points 2, 8, 10, 12, 26 and 28 hours N = 4; For time points 0, 4, 6, 24 and 30 hours N = 21. ** p<0.01 versus time point 0 hour. MiR-375 plasma levels (B) were measured in untreated mice (N = 6) and 4 (N = 3), 6 (N = 4) and 30 hours (N = 4) after injection of STZ. ** p<0.01 versus untreated mice.

Fig 2. Correlation between cytotoxicity assay and miR-375 release in culture supernatant of MIN-6 cells.

MIN-6 cells were incubated with different concentration of streptozotocin for 18 hours. Culture supernatant was collected and cytotoxicity assay (A) was performed on the remaining cells. Values are mean +/- SEM from three independent experiments performed in triplicate. **p<0.01 versus 0 mM STZ. MiR-375 analysis (B) was performed to evaluate miR-375 discharge. Values are mean +/- SEM from three independent experiments performed in triplicate. NS not significant; *p<0.05; **p<0.01 versus 0 mM STZ. Correlation between cytotoxicity and miR-375 release was calculated (C).

Fig 3. Glycemia and body weight in mice treated with STZ, PPAG or exendin-4.

Mice were treated with vehicle (CTRL), PPAG or exendin-4 (EX-4) at -48, -24, 0 and 24 hours. One injection of STZ or vehicle was given at time point 0 hour. Non-fasting glycemia (A) was measured at indicated time points (N = 10). *p<0.05; ***p<0.001 compared to CTRL. #p<0.05; ###p<0.001 compared to STZ. Body weight (% initial -48h weight) was measured (B) at indicated time points (N = 10). **p<0.01; ***p<0.001 compared to CTRL. ##p<0.01; ###p<0.001 Effect of 24 hours STZ.

Fig 4. Beta cell mass and apoptosis in mice treated with STZ alone, STZ + PPAG or STZ + exendin-4.

Mice were treated with vehicle (CTRL; N = 5), PPAG (N = 5) or exendin-4 (EX-4; N = 4) at -48, -24, 0 and 24 hours. One injection of STZ (N = 13) or vehicle (N = 5) was given at time point 0 hour. Thirty hours after STZ injection, beta cell mass (A) was measured. ***p<0.001 compared to CTRL mice. #p<0.05; ###p<0.001 Effect of PPAG and exendin-4 on STZ-induced decrease of beta cell mass. Beta cell apoptosis (B) was determined with TUNEL staining. *p<0.05; ***p<0.001 compared to CTRL mice. #p<0.05 Effect of PPAG and exendin-4 on STZ-induced increase of beta cell apoptosis.

Fig 5. MiR-375 in blood plasma of mice treated with STZ, STZ + PPAG or STZ + exendin-4.

Mice were treated with vehicle (CTRL; N = 15), PPAG (N = 12) or exendin-4 (EX-4; N = 8) at -48, -24, 0 and 24 hours. One injection of STZ (N = 35) or vehicle (N = 15) was given at time point 0 hour. Thirty hours after STZ injection, blood was collected and miR-375 was measured. ***p<0.001 compared to CTRL mice. ##p<0.01; ###p<0.001 Effect of PPAG and exendin-4 on STZ-induced increase of miR-375 levels.