Defective islet cell proliferation in Ran transgenic mice.

<p><i>A</i>, Pancreas section from non-TG or Ran-WT transgenic mice were harvested at the indicated postnatal (P) age and analyzed for Ki-67 reactivity, by immunohistochemistry. Sections from normal mouse spleen were used as control. <i>B</i>, The number of Ki-67⁺ cells/islet surface area (0.02 mm²) was quantified by morphometry. **, p = 0.0081. <i>C</i>, Pancreas section from non-TG or Ran-WT transgenic mice (P50) were analyzed for TUNEL reactivity and the numbers of positive cells was quantified. Mean±SD.</p

Characterization of Ran transgenic mice.

<p><i>A</i>, Tail DNA was extracted from transgenic mouse lines expressing Ran-WT, Ran T24N or Ran G19V in pancreatic β cells under RIP control, and PCR products were amplified using RIP-HA (<i>top</i>) or RIP-Ran (<i>bottom</i>) primers. (+) or (−) refers to Ran-positive or –negative genotype, or positive or negative control primers for the amplification reaction. M, molecular weight markers. <i>B</i>, Pancreas tissues extracted from the various mouse cohorts genotyped for the presence (+) or absence (−) of Ran transgenes were analyzed by Western blotting. 3T3, extracts from NIH3T3 cells transiently transfected with HA-Ran cDNA used as control. a-HA, antibody to HA. <i>C</i>: Pancreas or liver tissues were isolated from PCR-confirmed Ran transgenic mice, and analyzed by Western blotting. β-actin was used as a loading control.</p

Deregulated PDX-1 expression in Ran-targeted cells.

<p><i>A</i>, Pancreas sections from non-TG, Ran-WT, Ran-G19V or Ran-T24N transgenic mice were stained with an antibody to insulin or PDX-1, by immunohistochemistry. <i>B</i>, The number of insulin- or PDX-1-stained cells was quantified by morphometry in the indicated surface area. Insulin⁺ cells, non-TG (n = 2), 28.2±0.4; Ran-WT (asymptomatic, n = 3), 29.8±1.2; Ran-WT (diabetic, n = 3), 6.2±0.6, ***, p<0.0001; PDX-1⁺ cells, Non-TG (n = 2), 34.2±4.1; Ran-WT (asymptomatic, n = 3), 37.5±2.3; Ran-WT (diabetic, n = 3), 22.8±1.8, **, p = 0.007. <i>C</i>, Islets from 2 mo-old non-TG or a Ran-WT transgenic mouse were analyzed by Western blotting. <i>D</i>, Pancreatic islets isolated from non-TG mice were transduced <u>ex vivo</u> with control pAd-GFP or pAd-GFP-Ran-WT, pAd-GFP-Ran-G19V or pAd-GFP-T24N and analyzed after 48 h by fluorescence microscopy for GFP expression (<i>left</i>), or Western blotting (<i>right</i>). *, non-specific. <i>E</i>, INS-1 cells were left untreated (INS-1) or transduced with control lentivirus (pLKO) or lentivirus encoding Ran-directed shRNA (Ran, 74V1), and analyzed by Western blotting. <i>F</i>, Parental INS-1 cells or four independent clones of INS-1 cells stably transduced with Ran-directed shRNA (77V-2, 74V-1, 75V-1, 75V-2) were analyzed for changes in insulin release in the supernatant. Representative experiment out of at least two independent determinations.</p

Gender analysis of the diabetic phenotype in Ran transgenic mice.

<p>The indicated non-TG or Ran transgenic mice were analyzed for gender differences in blood glucose levels at 2 mo of age. Each point corresponds to an individual mouse.</p

Defective islet development in Ran transgenic mice.

<p><i>A</i>, Pancreas tissues from non-TG, Ran-WT, Ran-G19V or Ran-T24N transgenic mice were analyzed by H&E or immunohistochemical staining with an antibody to insulin. <i>B</i>, Pancreas sections from non-TG, asymptomatic Ran-WT or diabetic Ran-WT transgenic mice were analyzed for islet number or islet surface area by morphometry of insulin-stained areas. Islet number, non-TG (n = 2), 29.5±1.5; Ran-WT (n = 3), 23±1.73, n.s., not significant; Ran-WT diabetic (n = 3), 9.33±0.88, **, p = 0.001; islet surface area, non-TG (n = 2), 0.51±0.02; Ran-WT (n = 3), 0.35±0.021, *, p = 0.017; Ran-WT diabetic (n = 3), 0.057±0.004, **, p<0.0001. <i>C</i>, Pancreas tissues from Ran-WT, Ran-G19V or Ran-T24N transgenic mice were analyzed by immunohistochemical staining with antibodies to somatostatin or glucagon.</p

Transgenic expression of Ran impairs glucose metabolism.

<p><i>A and B</i>, The indicated PCR-confirmed transgenic mice were analyzed for blood glucose content at 2 mo of age under <u>ad libitum</u> feeding (<i>A</i>) or fasting (<i>B</i>) conditions. Non-TG, non-transgenic mice. Glucose concentrations (mg/dl) in each group in <i>A</i> (number of mice in parentheses), and statistical analyses (unpaired <i>t</i> test) are as follows: Non-TG (n = 24), 125±2; Ran-WT (n = 26), 189.4±17.8, p = 0.013; Ran-G19V (n = 24), 153.7±6.5, p = 0.003; Ran-T24N (n = 21), 172.4±21.1, p = 0.02. Statistical data re-analysis of the groups in <i>A</i> using ANOVA and post-hoc multiple tests with Bonferroni procedure was as follows: Ran-WT, p<0.0001; Ran-G19V, 0.017; Ran-T24N, p = 0.029. <i>C</i>, The indicated non-TG or Ran transgenic mice were analyzed for blood insulin concentrations. Insulin levels (ng/ml) in each group (number of mice in parentheses), and statistical analyses (unpaired <i>t</i> test) are as follows: Non-TG (n = 8), 1.26±0.1; Ran-WT (n = 11), 0.7±0.16, p = 0.019; Ran-G19V (n = 5), 0.61±0.03, p = 0.0008; Ran-T24N (n = 7), 0.49±0.14, p = 0.0009. Statistical data re-analysis using ANOVA and post-hoc multiple tests with Bonferroni procedure was as follows: Ran-WT, p = 0.023; Ran-G19V, p = 0.031; Ran-T24N, p = 0.004. One outlier mouse in the Ran-G19V group with aberrantly high insulin level (2.35 ng/ml) was excluded from the analysis. For panels <i>A–C</i>, each point corresponds to an individual mouse. <i>D</i>, Islets (20/well) isolated from non-TG or Ran-WT transgenic mice were incubated with 5 mM D-glucose, and analyzed for insulin release in the supernatant at the indicated time intervals. Mean±SD of replicates. <i>E</i>, Islets (20/well) isolated from non-TG (<i>black</i>) or Ran-WT (<i>purple</i>), Ran-G19V (<i>grey</i>) or Ran-T24N (<i>blue</i>) transgenic mice were incubated with 16.7 mM glucose, and analyzed for insulin release in the supernatant at the indicated time intervals. Mean±SD of replicates.</p