Quantitative RT-PCR analysis of imprinted genes.

<p>Expression of imprinted genes, <i>Rian</i> (A), <i>Plagl1</i> (B), <i>Dlk1</i> (C), and <i>Kcnq1</i> (D) in Pr-LP, Pr-HP, C4-LP, and C4-HP MIN6 cells. <i>Plagl1</i>, <i>Dlk1</i>, and <i>Rian</i> were confirmed to be responder genes, whereas <i>Kcnq1</i> was a non-responder gene. Values are means ± SD and n = 4–5. *<i>P</i><0.05.</p

Insulin secretion from MIN6 cells.

<p>Insulin secretion from Pr-LP, Pr-HP, C4-LP, and C4-HP MIN6 cells stimulated with 3 mM (3G), 8 mM (8G), 15 mM (15G), or 25 mM (25G) glucose (A, B), 100 nM glybenclamide (SU), or 30 mM KCl (C, D). Pr-HP cells showed higher basal insulin secretion at 3 mM glucose compared with Pr-LP cells, but their insulin secretion did not increase further at higher glucose concentrations or with the addition of glybenclamide or KCl, whereas both C4-LP and C4-HP MIN6 cells showed a better insulin secretory response to glucose and glybenclamide than Pr-LP cells. Values are means ± SD and n = 5–6. *<i>P</i><0.05 v.s. insulin secretion at 3 mM glucose.+<i>P</i><0.05 v.s. insulin secretion of Pr-LP, C4-LP, and C4-HP cells at 3 mM glucose by Student's <i>t</i>-test.</p

Microarray Analysis of Novel Candidate Genes Responsible for Glucose-Stimulated Insulin Secretion in Mouse Pancreatic β Cell Line MIN6

<div><p>Elucidating the regulation of glucose-stimulated insulin secretion (GSIS) in pancreatic islet β cells is important for understanding and treating diabetes. MIN6 cells, a transformed β-cell line derived from a mouse insulinoma, retain GSIS and are a popular <i>in vitro</i> model for insulin secretion. However, in long-term culture, MIN6 cells' GSIS capacity is lost. We previously isolated a subclone, MIN6 clone 4, from the parental MIN6 cells, that shows well-regulated insulin secretion in response to glucose, glybenclamide, and KCl, even after prolonged culture. To investigate the molecular mechanisms responsible for preserving GSIS in this subclone, we compared four groups of MIN6 cells: Pr-LP (parental MIN6, low passage number), Pr-HP (parental MIN6, high passage number), C4-LP (MIN6 clone 4, low passage number), and C4-HP (MIN6 clone 4, high passage number). Based on their capacity for GSIS, we designated the Pr-LP, C4-LP, and C4-HP cells as “responder cells.” In a DNA microarray analysis, we identified a group of genes with high expression in responder cells (“responder genes”), but extremely low expression in the Pr-HP cells. Another group of genes (“non-responder genes”) was expressed at high levels in the Pr-HP cells, but at extremely low levels in the responder cells. Some of the responder genes were involved in secretory machinery or glucose metabolism, including <i>Chrebp</i>, <i>Scgn</i>, and <i>Syt7</i>. Among the non-responder genes were <i>Car2</i>, <i>Maf</i>, and <i>Gcg</i>, which are not normally expressed in islet β cells. Interestingly, we found a disproportionate number of known imprinted genes among the responder genes. Our findings suggest that the global expression profiling of GSIS-competent and GSIS-incompetent MIN6 cells will help delineate the gene regulatory networks for insulin secretion.</p> </div

Insulin content and secretion of MIN6 cells.

<p>Insulin content of Pr-LP, Pr-HP, C4-LP, and C4-HP MIN6 cells (A). The insulin content of Pr-HP cells was lower than that of Pr-LP, C4-LP, or C4-HP cells. Values are means ± SD and n = 5–6. *<i>P</i><0.05. Insulin secretion/insulin content from Pr-LP, Pr-HP, C4-LP, and C4-HP MIN6 cells stimulated with 3 mM (3G), 25 mM (25G) glucose, 100 nM glybenclamide (SU), or 30 mM KCl (B, C). Values are means ± SD and n = 5–6. *<i>P</i><0.05 v.s. insulin secretion at 3 mM glucose.+<i>P</i><0.05 v.s. insulin secretion of Pr-LP, C4-LP, and C4-HP cells at 3 mM glucose by Student's <i>t</i>-test.</p

Expression levels of pancreas-related genes.

*<p>Raw values of expression intensities measured by Affymetrix arrays. **Mean value of C4-LP, C4-HP, and Pr-LP. ***Ratio of Pr-HP to mean value.</p

Genes preferentially expressed in non-responder MIN6 cells.

*<p>Raw values of expression intensities measured by Affymetrix arrays.</p

Analysis of CpG methylation of <i>Plagl1</i> and <i>Dlk1</i>.

<p>The ratio of methylated CpGs of each allele from Pr-LP, Pr-HP, C4-LP, and C4-HP MIN6 cells, in the <i>Plagl1</i> gene region (A) and the <i>Dlk1</i> gene region (B), by bisulfite sequencing. In the <i>Plagl1</i> gene locus, the pattern of methylation of CpG islands was quantitatively different between the responder MIN6 cells (Pr-LP, C4-LP, and C4-HP) and the non-responder cells (Pr-HP). In responder cells, one allele was either almost completely unmethylated and the other was almost completely methylated, whereas in the Pr-HP cells, both alleles were randomly methylated. In contrast, the <i>Dlk1</i> locus was significantly less methylated in Pr-HP cells than in the responder MIN6 cells. Values are means ± SD and n = 8-10. *<i>P</i><0.</p

Immunofluorescence analysis of the pancreas of RTF-Pdx1-EGFP mice given AdV-Isl1.

<p>(A)–(C) Pancreas was excised 1.5 days after AdV injection, and sections were stained with anti-CK (red), anti-phospho-STAT3 (light blue), and anti-Isl1 (green). Merged view of (A) and (B) is shown in (C) with DAPI (blue). Arrows indicate cells stained with both anti-Isl1 and anti-phospho-STAT3 antibodies. Bar = 100 µm. (D)–(F) Expression of MafA in the TC area. Sections from AdV-Isl1-injected RTF-Pdx1-EGFP mouse pancreas, excised 6 days after the injection, were stained with anti-insulin (red), anti-MafA (light blue), and anti-CK (green). Merged view of (D) and DAPI staining (blue) is shown in (E). Another part of the TC area is shown in (F) with DAPI staining. Heterogeneous staining for MafA was seen in the insulin-positive cells in the TC area. Arrows indicate insulin-positive, but MafA-negative cells. (G)–(J) Expression of MafA in the non-TC area of the same pancreatic section as (D–F). Merged view of (G) and DAPI staining is shown in (H). Merged view of (I) and DAPI staining is shown in (J). Almost all the insulin-positive islet cells were positive for MafA. Bars = 50 µm.</p

Immunofluorescence analysis of transdifferentiation in the AdV-Isl1-infected RTF-Pdx1-EGFP mouse pancreas.

<p>(A)–(D) Pancreas was excised 3 days after injection. Pancreatic sections were stained with anti-Ki67 (A, green) and anti-Isl1 (B, red). Merged view of (A) and (B) is shown in (C). Staining with anti-Isl1 (red) and DAPI (light blue) is shown in (D). Arrows indicate doubly stained cells. Note that most of the Ki67-positive cells were Isl1-positive. Bar = 100 µm. (E)–(G) Sections from AdV-Isl1-infected mouse pancreas, excised 3 days after injection, were stained with anti-CK (E, green) and anti-amylase (F, red). Merged view of (E) and (F) is shown in (G) with DAPI (blue). Arrows indicate CK and amylase double-positive cells. Bars = 50 µm. (H)–(J) Control pancreatic sections from wild-type mice not given an AdV injection were stained with anti-CK (H, green) and anti-amylase (I, red). Merged view of (H) and (I) is shown in (J) with DAPI (blue). Note that the duct was clearly stained with anti-CK but not with anti-amylase. Bars = 50 µm. (K)–(P) Pancreatic sections from AdV-Isl1-infected mice were stained with anti-CK (green), anti-Isl1 (magenta), and anti-amylase (red) with DAPI (blue). Merged image of (K) and (L) is shown in (M). Merged image of (N) and (O) is shown in (P). Arrows indicate CK/Isl1/amylase triple positive cells. Bar = 50 µm. (Q)–(S) Immunofluorescence analysis of the pancreas from mice given AdV-Isl1. Pancreas was excised 6 days after injection. Pancreatic sections were stained with anti-CK (green) and anti-Isl1 (magenta) with DAPI (blue) (Q) or with anti-amylase (red) and anti-Isl1 (magenta) (R). Merged view of (Q) and (R) is shown in (S). Note that most of the Isl1-positive cells in TCs were CK-positive. Bar = 50 µm.</p

The inducible Pdx1 mouse model.

<p>(A) The construct of the Tet-off regulation unit for Pdx1 expression that was integrated into the <i>ROSA26</i> locus. The tetracycline transactivator gene is expressed under the control of the ROSA26 promoter. In knock-in mice (RTF-Pdx1-EGFP mice) heterozygous for the transgene, the continuous administration of Dox prevents the tTA from binding to the tetO, thereby inactivating the transcription of Pdx1 cDNA. After Dox is withdrawn, tTA binds to the tetO, transcribing the Pdx1 cDNA and EGFP cDNA. (B)–(M) Immunofluorescence analysis of Pdx1 (red) and insulin (green) (B–G) or Pdx1 (red) and cytokeratin 19 (CK) (green) (H–M) in pancreas sections from RTF-Pdx1-EGFP mice treated with Dox or untreated for 17 days. Bars = 100 µm. (N)–(O) Magnified view of the area surrounded by the dotted line in (M). Staining with anti-CK antibody (green) and DAPI (4, 6-diamidino-2-phenylindole) (blue) is shown in (O). “d” and “i” represent a duct and an islet, respectively.</p