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. Author manuscript; available in PMC: 2014 Jun 15.
Published in final edited form as: Cell Rep. 2013 Sep 12;4(6):1262–1275. doi: 10.1016/j.celrep.2013.08.010

Figure 4. Nkx6.1 maintains Cyclin D2 expression and beta cell proliferative capacity through regulation of glucose uptake.

Figure 4

(A) qRT-PCR analysis of islets shows a decrease in Ccnd2 expression in Nkx6.1 Δadultβ compared to control mice at 6 wks (n=3). (B–C′) Immunofluorescence staining for insulin and Cyclin D2 shows a decrease in Cyclin D2 expression in beta cells of Nkx6.1 Δadultβ mice at 6 wks. B′ and C′ are higher magnification images of B and C, respectively. White arrowheads point to Cyclin D2high cells. (D) Immunoblot analysis of whole cell islet lysates confirms reduced Cyclin D2 expression in Nkx6.1 Δadultβ mice. (E) Quantification of the percentage of insulin+ cells expressing Ki67 shows decreased beta cell proliferation in Nkx6.1 Δadultβ mice at 6 wks (n=3). (F) qRT-PCR analysis of genes with decreased expression in islets from Nkx6.1 Δadultβ mice after adenoviral infection of Nkx6.1 Δadultβ islets with Ad-CMV-Glut2 (Ad-Glut2) and control islets with Ad-CMV-β-gal (Ad-β-gal) (n=3). Ad-Glut2 restores Ccnd2 expression to levels observed in control islets infected with Ad-β-gal. (G) Quantification of the percentage of insulin+ cells expressing Ki67 after infection of Nkx6.1 Δadultβ and control islets with Ad-Glut2 or Ad-β-gal shows that Ad-Glut2 restores the number of Ki67+ beta cells in Nkx6.1 Δadultβ islets to control values (n=3). (H) qRT-PCR analysis after a three-hour treatment of Nkx6.1 Δadultβ islets with the calcium channel activator Bay K8644 and control islets treated with vehicle (n=3). (I) Quantification of the percentage of insulin+ cells expressing Ki67 after injection of Nkx6.1 Δadultβ mice with 8mg/kg Bay K8644 or control mice injected with vehicle (n=3). Stimulation of calcium influx restores Ccnd2 expression and beta cell proliferation in Nkx6.1 Δadultβ mice. (J,K) qRT-PCR analysis of islets from Nkx6.1 Δadultβ mice treated with 10 μM glucokinase activator (GKA) (J) or 100 nM insulin (K) for 3 hours and islets from control mice treated with vehicle (n=3). Scale bar = 20 μm. Wk, week. Data are shown as mean ± SEM. *p<0.05, **p<0.01, ***p<0.001.