Beta-cell specific deletion of Dicer1 leads to defective insulin secretion and diabetes mellitus

Abstract

Mature microRNAs (miRNAs), derived through cleavage of pre-miRNAs by the Dicer1 enzyme, regulate protein expression in many cell-types including cells in the pancreatic islets of Langerhans. To investigate the importance of miRNAs in mouse insulin secreting β-cells, we have generated mice with a β-cells specific disruption of the Dicer1 gene using the Cre-lox system controlled by the rat insulin promoter (RIP). In contrast to their normoglycaemic control littermates (RIP-Cre(+/-) Dicer1(Δ/wt)), RIP-Cre(+/-)Dicer1(flox/flox) mice (RIP-Cre Dicer1(Δ/Δ)) developed progressive hyperglycaemia and full-blown diabetes mellitus in adulthood that recapitulated the natural history of the spontaneous disease in mice. Reduced insulin gene expression and concomitant reduced insulin secretion preceded the hyperglycaemic state and diabetes development. Immunohistochemical, flow cytometric and ultrastructural analyses revealed altered islet morphology, marked decreased β-cell mass, reduced numbers of granules within the β-cells and reduced granule docking in adult RIP-Cre Dicer1(Δ/Δ) mice. β-cell specific Dicer1 deletion did not appear to disrupt fetal and neonatal β-cell development as 2-week old RIP-Cre Dicer1(Δ/Δ) mice showed ultrastructurally normal β-cells and intact insulin secretion. In conclusion, we have demonstrated that a β-cell specific disruption of the miRNAs network, although allowing for apparently normal β-cell development, leads to progressive impairment of insulin secretion, glucose homeostasis and diabetes development.

Figure 1. RIP-Cre Dicer1^Δ/Δ mice develop diabetes during the first 25 weeks of age.

A. PCR analysis of tail genomic DNA showing the genotypes of the mice used in the experiments, lane 1 RIP-Cre Dicer1^Δ/Δ and lane 2 their control littermates RIP-Cre Dicer1^{Δ /wt} B. PCR analysis of genomic DNA from sorted β-cells demonstrating deletion of both alleles of Dicer1 gene in RIP-Cre Dicer1^Δ/Δ and deletion of only one allele in control littermates RIP-Cre Dicer1^{Δ /wt} mice. Numbers below lanes represent the two PCR reactions (one for each allele) for each mouse strain. C. Cumulative diabetes incidence in RIP-Cre Dicer1^Δ/Δ mice. Glucose was measured every week from the age of 3 weeks using a glucometer. Control littermates never develop diabetes or hyperglycaemia and are therefore not shown in the figure. D. Plasma blood glucose levels (mmol/l) measured in 4, 7 and 13 weeks old RIP-Cre Dicer1^Δ/Δ mice and littermate controls as indicated. The number of animals within the different groups is indicated below. Data are presented as mean±sd. ***P<0.001.

Figure 2. Impaired in vivo glucose tolerance and in vitro insulin secretion in RIP-Cre Dicer1^Δ/Δ mice.

A. In vivo glucose tolerance test was performed on 6 control littermates and 5 RIP-Cre Dicer1^Δ/Δ females at 4–5 weeks of age (left) and 6 control littermates and 6 RIP-Cre Dicer1^Δ/Δ females at 13 weeks of age (right). Note that the glucose levels of the 13 weeks old RIP-Cre Dicer1^Δ/Δ mice reached levels not measurable by the glucometer. Data are mean±SEM. B. Insulin secretion from isolated pancreatic islets was measured after 1 h incubation in 1 mM glucose (1 G) or 20 mM glucose (20 G) in 2 and 5–6 weeks old littermate control (white bar) or RIP-Cre Dicer1^Δ/Δ mice (black bar). C. (Left) As in B, but insulin secretion was measured in 7–8 weeks old mice. (Right) Insulin release normalized to insulin content. Data are mean±SEM of 15–21 experiments in 3–5 animals. *P<0.05; ***P<0.001.

Figure 3. Altered islet morphology in RIP-Cre Dicer1^Δ/Δ mice.

H-E stainings of 5, 8, 11 and 25 weeks old littermate controls (left) or RIP-Cre Dicer1^Δ/Δ (right) mice. Representative islets of more than 5 mice per group and age are shown. All images are 20× magnification.

Figure 4. Altered cellular morphology in RIP-Cre Dicer1^Δ/Δ islets with increasing age.

Confocal microscopy analysis of insulin and glucagon staining was performed on islets from 5, 8 and 11 weeks old control littermates (upper panels) and RIP-Cre Dicer1^Δ/Δ (lower panels) mice. Insulin and glucagon staining can be observed as indicated. Notice the deformation of the RIP-Cre Dicer1^Δ/Δ islet and the increased number of α-cells relative to the reduced number of β-cells. Scale bar 20 µm.

Figure 5. Decreased insulin-producing cells in RIP-Cre Dicer1^Δ/Δ islets.

Islet cells suspension from 50 islets from 8 weeks old littermates and RIP-Cre Dicer1^Δ/Δ mice were stained for insulin and glucagon and analyzed by flow cytometry. Side scatter (SSC) and forward scatter (FSC) analysis of islet cells and the respective gate used to identify live cells. Littermate control islet cells contained significantly more insulin-positive cells (P<0.001) and less glucagon-positive cells (P<0.05) than the RIP-Cre Dicer1^Δ/Δ islet cells. Representative of 3 independent experiments.

Figure 6. Decreased total β-cell mass in RIP-Cre Dicer1^Δ/Δ visualised by optical projection tomography.

A. Optical tomographic sections based on insulin-labeling of adult pancreas of 5, 8 and 12 weeks old RIP-Cre Dicer1 and control littermate mice. A clear decrease of total β-cells can be observed in pancreas from 8 and 12 weeks old hyperglycemic in RIP-Cre Dicer1^Δ/Δ mice. Notice that the image shown is a 2D image and that the OPT technique generate a 3D reconstruction of the whole pancreas. B. Quantification of β-cell volume per pancreas volume from 5, 8 and 12 week old RIP-Cre Dicer1 and control littermate mice. The number of animals used in the analysis of the different groups is given below the columns. Data are presented as mean±SEM for the 8 and 12 week old mice. *P<0.05.

Figure 7. Ultrastructural changes in RIP-Cre Dicer1^Δ/Δ β-cells.

A. Electron micrograph of β-cells from 11 weeks old control (top) and RIP-Cre Dicer1^Δ/Δ (bottom) mice. In the left images (low magnification) the plasma membrane surrounding the analyzed β-cell is marked by a black line. The area within the dotted rectangle is shown in the higher magnification images to the right. Docked granules are marked with black arrows in the images to the right. Scale bars 2 µm (left images) and 0.5 µm (right images). β: β-cell; α: α-cell; δ: δ-cell; N: nucleus; g: granule; m:mitochondria. B. Histograms of the total amount of granules (left) and the number of docked granules (right) within β-cells measured as volume density N_V (granules/µm³) and surface density N_s (granules/µm²), respectively. Analysis was performed on β-cells within islets from 11 weeks old control littermates (white bar) and RIP-Cre Dicer1^Δ/Δ mice (black bar). Data is expressed as mean±SEM of 23 cells in each group. ***P<0.001. The embedded islets where from N = 4 mice. C. As in B, but analysis was performed on β-cells within islets from 2 week old control littermates (white bar) and RIP-Cre Dicer1^Δ/Δ mice (black bar). Data are from N = 2 mice each for control and RIP-Cre Dicer1^Δ/Δ mice, and the mean from each of the individual mice are presented in the graph.

Figure 8. Insulin, miR-375 and pri-miR-375 expression in sorted β-cells.

Pancreatic islets were isolated from 7 RIP-Cre Dicer1^Δ/Δ and 7 littermate control mice. β-cells were sorted using flow cytometry to >98% purity. Total RNA was extracted and used for qPCR and array hybridization. A. Insulin mRNA expression level is reduced in RIP-Cre Dicer1^Δ/Δ, B. Mature miR-375 is significantly reduced while the pri-miR-375 species accumulates in the knockout. ***P<0.001.

Figure 9. No evidence of increased β-cell apoptosis and altered proliferation in RIP-Cre Dicer1^Δ/Δ mice.

A. β-cell apoptosis was estimated using in situ TUNEL assay from 8–12 weeks old RIP-Cre Dicer1^Δ/Δ and littermate control mice (right). No significant apoptosis could be detected in any islets observed. Nuclease-treated pancreas section from age matched littermate control mice was used as positive control for TUNEL staining and is depicted on the left. These are representative of several sections of individual pancreases. B. β-cell proliferation was detected in situ based on Ki67 expression and analysed by confocal microscopy in 11 weeks old mice. Upper panels depict insulin and Ki67 staining in control littermates whereas the lower panels represent RIP-Cre Dicer1^Δ/Δ pancreas. Although RIP-Cre Dicer1^Δ/Δ islets have a clear decrease of insulin-producing cells compared to control littermates, no sign of increased proliferating Ki67 positive cells is observed.