miR-375 gene dosage in pancreatic β-cells: implications for regulation of β-cell mass and biomarker development

Abstract

MicroRNAs play a crucial role in the regulation of cell growth and differentiation. Mice with genetic deletion of miR-375 exhibit impaired glycemic control due to decreased β-cell and increased α-cell mass and function. The relative importance of these processes for the overall phenotype of miR-375KO mice is unknown. Here, we show that mice overexpressing miR-375 exhibit normal β-cell mass and function. Selective re-expression of miR-375 in β-cells of miR-375KO mice normalizes both, α- and β-cell phenotypes as well as glucose metabolism. Using this model, we also analyzed the contribution of β-cells to the total plasma miR-375 levels. Only a small proportion (≈1 %) of circulating miR-375 originates from β-cells. Furthermore, acute and profound β-cell destruction is sufficient to detect elevations of miR-375 levels in the blood. These findings are supported by higher miR-375 levels in the circulation of type 1 diabetes (T1D) subjects but not mature onset diabetes of the young (MODY) and type 2 diabetes (T2D) patients. Together, our data support an essential role for miR-375 in the maintenance of β-cell mass and provide in vivo evidence for release of miRNAs from pancreatic β-cells. The small contribution of β-cells to total plasma miR-375 levels make this miRNA an unlikely biomarker for β-cell function but suggests a utility for the detection of acute β-cell death for autoimmune diabetes.

Key messages: • Overexpression of miR-375 in β-cells does not influence β-cell mass and function. • Increased α-cell mass in miR-375KO arises secondarily to loss of miR-375 in β-cells. • Only a small proportion of circulating miR-375 levels originates from β-cells. • Acute β-cell destruction results in measurable increases of miR-375 in the blood. Circulating miR-375 levels are not a biomarker for pancreatic β-cell function.

Keywords: Biomarker; Diabetes; MiRNA-375; Pancreatic β-cells; β-cell mass.

Fig. 1

Metabolic characterization of β-cell-specific miR-375 transgenic mice. a Relative miR-375 expression in islets and indicated organs of male wildtype (WT) and Tg375 mice at 12 weeks of age (n = 5 for islets, n = 2 for all other tissues). b Relative expression of validated miR-375 targets in islets of male Tg375 mice and WT controls at 12 week of age. c Body weight of Tg375 (white circles) and WT (black circles) mice (n = 10). d Ad libitum-fed blood glucose levels in Tg375 and control littermate mice (n = 10). e Intraperitoneal Glucose Tolerance Test (IPGTT; 2 g/kg) in overnight fasted Tg375 (white circles) and WT (black circles) mice at 10 weeks of age (n = 11). f Static insulin secretion performed with 10-week-old control and Tg375 islets (n = 5) at 3.3 mmol/l (white bars) and 16.7 mmol/l (black bars) glucose concentrations. All data shown are mean ± s.e.m., except for panel c and d where s.d. is shown. *p < 0.05, **p < 0.01

Fig. 2

Functional characterization of miR-375KO mice with selective re-expression of miR-375 in pancreatic β-cells. a Relative miR-375 expression in islets of male WT, Tg375, miR-375KO, and β-Rescue mice at 12 weeks of age (n = 4–5). Data expressed as fold-change over WT controls. b Relative expression of the miR-375 target HuD in islets of male WT, Tg375, miR-375KO, and β-Rescue mice at 12 weeks of age (n = 4–5). c Detection of miR-375 and 28S rRNA in pancreatic tissue sections from wildtype (WT), miR-375KO, and β-Rescue mice using miRNA FISH. Green: miR-375, Red: 28S rRNA, Blue: cell nuclei. All data shown are mean ± s.e.m. *p < 0.05, ***p < 0.005

Fig. 3

Metabolic characterization of miR-375 β-Rescue mice. WT, Tg375, miR-375KO, and β-Rescue mice were subjected to the following measurements: a body weight, b ad libitum-fed blood glucose levels, c fasting glucose (6 h and overnight) levels at 12 weeks of age, d IPGTT (2 g/kg) after an overnight fast at 13 weeks of age, e ITT (0.75U/kg) after an overnight fast at 11 weeks of age, f insulin excursion analysis after an overnight fast at 16 weeks of age (3 g/kg). All data shown are mean ± s.e.m; n = 6–9; **p < 0.01, ***p < 0.005 WT vs. miR-375KO, ^# p < 0.05, ^## p < 0.01, β-Rescue vs. miR-375KO

Fig. 4

Hormone levels and islet cell mass in miR-375 β-Rescue mice. WT, Tg375, miR-375KO, and β-Rescue mice were subjected to the following measurements after a 6-h fast: a circulating plasma insulin levels and b pancreatic insulin content at 24 weeks of age (n = 5–6), c normalized pancreatic α-cell at 5 weeks of age (n = 5–7), d plasma glucagon levels in mice fasted for 3 h at 24 weeks of age (n = 5–6), e pancreatic β-cell mass at 5 weeks of age (n = 3–5), f intraperitoneal PTT in mice fasted overnight at 15 weeks of age (2 g/kg) (n = 6–9). All data shown are mean ± s.e.m; *p < 0.05, **p < 0.01, ***p < 0.005 WT vs. miR-375KO, ^# p < 0.05, ^## p < 0.01, ^### p < 0.005, β-Rescue versus miR-375KO

Fig. 5

Pancreatic islets secrete miR-375 in the circulation. a Detection of miRNAs in C57BL/6 mouse plasma. Shown is the mean Ct value of indicated miRNAs evaluated by qPCR after 45 cycles (n = 3). b Copy number of circulating miR-375 and miR-16 in C57BL/6 mice at 7 weeks. c, d miR-375 copy number per ml of plasma of wildtype (WT), Tg375, miR-375KO, and β-Rescue mice at 20 weeks of age. e Relative miR-375 levels in supernatant of pancreatic islets isolated from WT, miR-375KO, and Tg375 mice cultured in serum-free media for 16 h at 37 °C. Media, serves as a negative control (n = 3). Data expressed as fold-change over WT controls. All data shown are mean ± s.e.m; *p < 0.05, ***p < 0.005

Fig. 6

Correlation between circulating miR-375 levels and β-cell injury. a Blood glucose levels and b pancreatic insulin content in 6-h fasted C57BL/6 (10-week-old) after 72 h treatment with streptozotocin (STZ, 1 × 150 mg/kg) (n = 7–8). c Circulating miR-375 and d miR-16 copy number in plasma of 6-h fasted C57BL/6 WT mice (10-week-old) after being injected with STZ (+, 1 × 150 mg/kg) or PBS as control (−) for 3 days (n = 7–8). e Blood glucose and f circulating miR-375 and g miR-16 levels in WT and db/db (BKS-background) male mice at 8 weeks of age (n = 4–5). h Blood glucose, i pancreatic insulin content, and j circulating miR-375 and k miR-16 in C57BL/6 (WT) mice fed a normal or high-fat diet (HFD, for 25 weeks) and ob/ob (C57BL/6 background) mice (23-week-old) (n = 5). l Circulating miR-375 levels in healthy or no diagnosed metabolic disease patients (n = 51), HNF1α/MODY3 mutation carriers (n = 47), T1D (n = 38) and T2D (n = 58). All data shown are mean ± s.e.m; *p < 0.05, **p < 0.01, ***p < 0.005