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Generation of insulin-producing cells from the mouse liver using β cell-related gene transfer including Mafa and Mafb.

Nagasaki H, Katsumata T, Oishi H, Tai PH, Sekiguchi Y, Koshida R, Jung Y, Kudo T, Takahashi S - PLoS ONE (2014)

Bottom Line: 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.They also show that Mafa has a markedly intense and sustained role in β-like cell production in comparison with Mafb.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Embryology, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan.

ABSTRACT
Recent studies on the large Maf transcription factors have shown that Mafb and Mafa 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 Mafb before birth and of Mafa 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 in vivo 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 Pdx1/Neurod/Mafa (PDA) and Pdx1/Neurod/Mafb (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 Mafa has a more sustainable role in insulin gene activation than does Mafb. 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 Mafa has a markedly intense and sustained role in β-like cell production in comparison with Mafb.

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Comparison of the transcription of insulin in the liver by use of bioluminescence imaging.(A) Representative bioluminescence imaging of MIP-Luc-VU mice after Pdx1/Neurod (PD), Pdx1/Neurod/Mafa (PDA), and Pdx1/Neurod/Mafb (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.
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pone-0113022-g002: Comparison of the transcription of insulin in the liver by use of bioluminescence imaging.(A) Representative bioluminescence imaging of MIP-Luc-VU mice after Pdx1/Neurod (PD), Pdx1/Neurod/Mafa (PDA), and Pdx1/Neurod/Mafb (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.

Mentions: To compare the effects of Mafa and Mafb on the transcriptional activity of the insulin gene through measurement of the bioluminescence emission, we examined 3 different gene combinations – Pdx1/Neurod (PD); Pdx1, Neurod, and Mafa (PDA); and Pdx1, Neurod, and Mafb (PDB) – in MIP-Luc-VU mice (Figure 2A). The bioluminescence signals induced by the 3 different combinations peaked at day 3 and gradually disappeared over 14 days in the same way. The PDA (1.81±0.34×109/photons/sec/cm2/steradian; n = 7) and PDB (1.70±0.33×109/photons/sec/cm2/steradian; n = 8) transductions induced comparable but significantly higher signal intensities than the peak emission of PD (4.49±2.6×107/photons/sec/cm2/steradian; n = 4) at day 3 (Figure 2B). In contrast, only the PDA-transferred mice continued to emit substantial bioluminescence, even at day 10, suggesting that Mafa is capable of inducing a peak emission similar to but a more sustainable insulin gene activity than that of Mafb (PD: 1.28±0.1×105/photons/sec/cm2/steradian; n = 4, PDA: 2.46±0.8×107/photons/sec/cm2/steradian; n = 7, PDB: 1.96±0.4×105/photons/sec/cm2/steradian; n = 8) (Figure 2C). Furthermore, immunohistochemistry using anti-insulin antibody revealed that some PDA- and PDB-transferred liver cells reacted to the antibody at days 3 and 9 after infection (Figure 2D). To confirm the presence of insulin-positive cells in PDB-transferred liver, DAB (3, 3′-diaminobenzidine) visualization is also shown in Figure S2A. We next examined the insulin content of the gene-transferred livers at days 3 and 9 after infection (Figure 3). Consistent with the bioluminescence results, we found no difference among the groups at day 3 (GFP: 0.31±0.2 ng/mg; n = 4; PDA: 1.26±0.5 ng/mg; n = 7; PDB: 2.6±0.3 ng/mg; n = 5), but the PDA-transferred liver at day 9 (6.23±1.3 ng/mg, n = 4) contained fairly abundant amounts of insulin protein when compared with the other groups. Interestingly, the insulin content of the PDA-transferred liver at day 9 was increased 5-fold over that of day 3, suggesting that newly synthesized insulin protein is gradually stored in the liver.


Generation of insulin-producing cells from the mouse liver using β cell-related gene transfer including Mafa and Mafb.

Nagasaki H, Katsumata T, Oishi H, Tai PH, Sekiguchi Y, Koshida R, Jung Y, Kudo T, Takahashi S - PLoS ONE (2014)

Comparison of the transcription of insulin in the liver by use of bioluminescence imaging.(A) Representative bioluminescence imaging of MIP-Luc-VU mice after Pdx1/Neurod (PD), Pdx1/Neurod/Mafa (PDA), and Pdx1/Neurod/Mafb (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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4232560&req=5

pone-0113022-g002: Comparison of the transcription of insulin in the liver by use of bioluminescence imaging.(A) Representative bioluminescence imaging of MIP-Luc-VU mice after Pdx1/Neurod (PD), Pdx1/Neurod/Mafa (PDA), and Pdx1/Neurod/Mafb (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.
Mentions: To compare the effects of Mafa and Mafb on the transcriptional activity of the insulin gene through measurement of the bioluminescence emission, we examined 3 different gene combinations – Pdx1/Neurod (PD); Pdx1, Neurod, and Mafa (PDA); and Pdx1, Neurod, and Mafb (PDB) – in MIP-Luc-VU mice (Figure 2A). The bioluminescence signals induced by the 3 different combinations peaked at day 3 and gradually disappeared over 14 days in the same way. The PDA (1.81±0.34×109/photons/sec/cm2/steradian; n = 7) and PDB (1.70±0.33×109/photons/sec/cm2/steradian; n = 8) transductions induced comparable but significantly higher signal intensities than the peak emission of PD (4.49±2.6×107/photons/sec/cm2/steradian; n = 4) at day 3 (Figure 2B). In contrast, only the PDA-transferred mice continued to emit substantial bioluminescence, even at day 10, suggesting that Mafa is capable of inducing a peak emission similar to but a more sustainable insulin gene activity than that of Mafb (PD: 1.28±0.1×105/photons/sec/cm2/steradian; n = 4, PDA: 2.46±0.8×107/photons/sec/cm2/steradian; n = 7, PDB: 1.96±0.4×105/photons/sec/cm2/steradian; n = 8) (Figure 2C). Furthermore, immunohistochemistry using anti-insulin antibody revealed that some PDA- and PDB-transferred liver cells reacted to the antibody at days 3 and 9 after infection (Figure 2D). To confirm the presence of insulin-positive cells in PDB-transferred liver, DAB (3, 3′-diaminobenzidine) visualization is also shown in Figure S2A. We next examined the insulin content of the gene-transferred livers at days 3 and 9 after infection (Figure 3). Consistent with the bioluminescence results, we found no difference among the groups at day 3 (GFP: 0.31±0.2 ng/mg; n = 4; PDA: 1.26±0.5 ng/mg; n = 7; PDB: 2.6±0.3 ng/mg; n = 5), but the PDA-transferred liver at day 9 (6.23±1.3 ng/mg, n = 4) contained fairly abundant amounts of insulin protein when compared with the other groups. Interestingly, the insulin content of the PDA-transferred liver at day 9 was increased 5-fold over that of day 3, suggesting that newly synthesized insulin protein is gradually stored in the liver.

Bottom Line: 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.They also show that Mafa has a markedly intense and sustained role in β-like cell production in comparison with Mafb.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Embryology, Faculty of Medicine, University of Tsukuba, Tsukuba, Ibaraki, Japan.

ABSTRACT
Recent studies on the large Maf transcription factors have shown that Mafb and Mafa 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 Mafb before birth and of Mafa 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 in vivo 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 Pdx1/Neurod/Mafa (PDA) and Pdx1/Neurod/Mafb (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 Mafa has a more sustainable role in insulin gene activation than does Mafb. 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 Mafa has a markedly intense and sustained role in β-like cell production in comparison with Mafb.

Show MeSH
Related in: MedlinePlus