Generation of Insulin-Producing Cells from the Mouse Liver Using β Cell-Related Gene Transfer Including <i>Mafa</i> and <i>Mafb</i>

<div><p>Recent studies on the large Maf transcription factors have shown that <i>Mafb</i> and <i>Mafa</i> have respective and distinctive roles in β-cell development and maturation. However, whether this difference in roles is due to the timing of the gene expression (roughly, expression of <i>Mafb</i> before birth and of <i>Mafa</i> after birth) or to the specific function of each gene is unclear. Our aim was to examine the functional differences between these genes that are closely related to β cells by using an <i>in vivo</i> model of β-like cell generation. We monitored insulin gene transcription by measuring bioluminescence emitted from the liver of insulin promoter-luciferase transgenic (MIP-Luc-VU) mice. Adenoviral gene transfers of <i>Pdx1/Neurod/Mafa</i> (PDA) and <i>Pdx1/Neurod/Mafb</i> (PDB) combinations generated intense luminescence from the liver that lasted for more than 1 week and peaked at 3 days after transduction. The peak signal intensities of PDA and PDB were comparable. However, PDA but not PDB transfer resulted in significant bioluminescence on day 10, suggesting that <i>Mafa</i> has a more sustainable role in insulin gene activation than does <i>Mafb</i>. Both PDA and PDB transfers ameliorated the glucose levels in a streptozotocin (STZ)-induced diabetic model for up to 21 days and 7 days, respectively. Furthermore, PDA transfer induced several gene expressions necessary for glucose sensing and insulin secretion in the liver on day 9. However, a glucose tolerance test and liver perfusion experiment did not show glucose-stimulated insulin secretion from intrahepatic β-like cells. These results demonstrate that bioluminescence imaging in MIP-Luc-VU mice provides a noninvasive means of detecting β-like cells in the liver. They also show that <i>Mafa</i> has a markedly intense and sustained role in β-like cell production in comparison with <i>Mafb</i>.</p></div

Bioluminescence emission from the hepatic region of MIP-Luc-VU mice after β cell-related gene transfer.

<p>(A) Diagrammatic representation of bioluminescence monitoring of insulin transcriptional activity. (B) Representative example of dose-dependent bioluminescence emission from the hepatic region. (C) Bioluminescence images of the abdominal section of MIP-Luc-VU mice 3 days after infection. (D) Tissue sections of MIP-Luc-VU liver stained with anti-luciferase antibody with 4′,6-diamidino-2-phenylindole (DAPI) 3 days after gene transfer. Scale bar indicates 100 µm.</p

Primer sequences used in the study.

<p>Primer sequences used in the study.</p

Comparison of the transcription of insulin in the liver by use of bioluminescence imaging.

<p>(A) Representative bioluminescence imaging of MIP-Luc-VU mice after <i>Pdx1</i>/<i>Neurod</i> (PD), <i>Pdx1</i>/<i>Neurod</i>/<i>Mafa</i> (PDA), and <i>Pdx1</i>/<i>Neurod</i>/<i>Mafb</i> (PDB) gene transfer. (B, C) Quantification of signal intensity after PD (n = 6), PDA (n = 7), and PDB (n = 7) gene transfer at days 3 (B) and 10 (C). (D) Tissue sections of wild-type mouse liver stained with anti-insulin antibody (red) and 4′6-diamidino-2-phenylindole (DAPI) (blue) after GFP-, PDA-, and PDB-gene transfer. Arrowheads indicate insulin-positive cells. Scale bars indicate 100 µm.</p

Expression of β cell-related genes in mouse liver after <i>Pdx1/Neurod</i> in combination with <i>Mafa</i> (PDA) or <i>Mafb</i> (PDB) gene transfer.

<p>Wild-type mouse liver treated with Ad-PDA (PDA, n = 7) displayed a significant increase in β cell-related mRNAs at day 9 (<i>Ins1</i>, <i>Ins2</i>, <i>PC2</i>, <i>i-GK</i>, <i>Sur1</i>, <i>Kir6.2</i>) on Q-PCR analysis. In contrast, the Ad-PDB treated group of mice (PDB, n = 7) displayed a significant increase in <i>Ins1</i>, <i>Ins2</i>, <i>Sur1</i>, and <i>Kir6.2</i> only at day 3. Data were normalized to <i>Hprt</i> mRNA abundance and shown as relative expression levels to that of the Ad-PDA-treated group at day 3. Data were expressed as the means ± standard errors of the means and analyzed using the Steel-Dwass multiple comparison test.</p

Glucose-independent insulin release from the mouse liver after β-like cell-related gene transfer.

<p>(A–B) Glucose-stimulated insulin release from the livers of Ad-GFP- (n = 6), Ad-PDA- (n = 6), and Ad-PDB- (n = 7) treated mice 7 days (A) and 14 days (B) after treatment by <i>in situ</i> liver perfusion. Data were expressed as the means ± standard errors of the means, and <i>P</i> values calculated using the Tukey-Kramer honestly significant difference (HSD) test. *<i>P</i><0.05. N.D. indicates no data.</p

Insulin content in mouse liver after β-like cell-related gene transfer.

<p>Insulin content of wild-type mouse liver at days 3 and 9 after GFP (n = 7), <i>Pdx1</i>/<i>Neurod</i>/<i>Mafa</i> (PDA, n = 8), and <i>Pdx1</i>/<i>Neurod</i>/<i>Mafb</i> (PDB, n = 5) gene transfer. Data were expressed as the means ± standard errors of the means and analyzed by one-way ANOVA followed by the Tukey-Kramer honestly significant difference (HSD) test.</p