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 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

Transgenic Expression of a Single Transcription Factor Pdx1 Induces Transdifferentiation of Pancreatic Acinar Cells to Endocrine Cells in Adult Mice

<div><p>A promising approach to new diabetes therapies is to generate β cells from other differentiated pancreatic cells <i>in vivo</i>. Because the acinar cells represent the most abundant cell type in the pancreas, an attractive possibility is to reprogram acinar cells into β cells. The transcription factor Pdx1 (Pancreas/duodenum homeobox protein 1) is essential for pancreatic development and cell lineage determination. Our objective is to examine whether exogenous expression of Pdx1 in acinar cells of adult mice might induce reprogramming of acinar cells into β cells. We established a transgenic mouse line in which Pdx1 and EGFP (enhanced green fluorescent protein) could be inducibly expressed in the acinar cells. After induction of Pdx1, we followed the acinar cells for their expression of exocrine and endocrine markers using cell-lineage tracing with EGFP. The acinar cell-specific expression of Pdx1 in adult mice reprogrammed the acinar cells as endocrine precursor cells, which migrated into the pancreatic islets and differentiated into insulin-, somatostatin-, or PP (pancreatic polypeptide)-producing endocrine cells, but not into glucagon-producing cells. When the mice undergoing such pancreatic reprogramming were treated with streptozotocin (STZ), the newly generated insulin-producing cells were able to ameliorate STZ-induced diabetes. This paradigm of <i>in vivo</i> reprogramming indicates that acinar cells hold promise as a source for new islet cells in regenerative therapies for diabetes.</p></div

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 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

Immunohistochemical analysis of the pancreas of ERTF-Pdx1-EGFP mice maintained without Dox.

<p>Sections from the pancreas of ERTF-Pdx1-EGFP mice 4, 7, and 10 weeks after Dox withdrawal (<i>n</i> = 3, 3, and 4, respectively) were stained for insulin, EGFP, and DNA. (A) The percentage of EGFP-positive cells among insulin-positive islet cells was calculated for each mouse. The total number of insulin-positive cells examined was 4968, 6690, and 9555 at 4, 7, and 10 weeks after Dox withdrawal, respectively. Statistical analysis was performed by one-way ANOVA followed by Tukey’s post-hoc test. *<i>P</i> < 0.05. (B) EGFP expression in islet-like clusters containing fewer than 10 insulin-positive cells was examined for the pancreas of these mice. The percentage of islet-like clusters containing insulin-producing cells with only EGFP-negative (open bars), EGFP-positive and negative (grey bars), and only EGFP-positive (black bars) cells was determined. (C-E) Immunohistochemical analysis of the pancreas of ERTF-Pdx1-EGFP mice 10 weeks after Dox withdrawal. Sections were stained for insulin (red) and EGFP (green). Right panel (E) is a merged view of (C) and (D). Arrow shows an islet-like cluster in which all the insulin-positive cells were also EGFP-positive. Arrowhead shows EGFP-positive insulin-producing cells in an islet-like cluster. Bar = 50 μm. (F-I) Increased BrdU-positive cells in the islets of ERTF-Pdx1-EGFP mice 6 weeks after Dox withdrawal. Pancreas sections were stained for BrdU (red), DNA (blue), and EGFP (green). Merged image of (G) with insulin staining (blue) is shown in (H). Bar = 25 μm. The percentage of BrdU-positive cells among EGFP-negative and -positive insulin-positive islet cells was determined (I). In the islets, BrdU-positive cells were significantly more enriched in EGFP/insulin double-positive cells than in insulin-positive EGFP-negative cells. The total number of insulin-positive islet cells examined was 1514, 1762, 2233, and 3375 each from four ERTF-Pdx1-EGFP mice. **<i>P</i> < 0.01. (J-L) Pancreas sections from ERTF-Pdx1-EGFP mice 10 weeks after Dox withdrawal were stained for glucagon (red) and EGFP (green). Serial section to that used in (C-E) was used. Right panel (L) is a merged view of (J) and (K). No EGFP/glucagon double-positive cells were observed. Bar = 50 μm.</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

Immunohistochemical analysis of the pancreas of STZ-treated ERTF-Pdx1-EGFP mice without Dox.

<p>ERTF-Pdx1-EGFP mice were given STZ 4 weeks after Dox withdrawal. Six weeks after STZ injection, their pancreata were used for immunostaining analyses. (A) Immunostaining for insulin (red) and EGFP (green). Bar = 200 μm. (B-G) Magnified views of the islet regions including the dotted line-box in (A). Bars = 25 μm. Merged images including DAPI staining are also shown in (D) and (G). (H) Insulin-positive cells were rarely seen in the STZ-treated wild-type controls. (I) The percentage of EGFP-positive cells among insulin-positive islet cells was determined for each islet of the pancreas from three STZ-treated and three STZ-untreated ERTF-Pdx1-EGFP mice, 10 weeks after Dox withdrawal (<i>n</i> = 18–40 for each mouse). Each bar represents the mean for a single animal (19%, 28%, 24%, 63%, 60%, and 69%, respectively). The proportion of EGFP-positive cells among insulin-positive islet cells was significantly higher in the STZ-treated animals than in the STZ-untreated controls (<i>P</i> < 0.05).</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