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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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Bioluminescence emission from the hepatic region of MIP-Luc-VU mice after β cell-related gene transfer.(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.
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pone-0113022-g001: Bioluminescence emission from the hepatic region of MIP-Luc-VU mice after β cell-related gene transfer.(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.

Mentions: To screen the transcriptional activity of the intrahepatic insulin gene in a noninvasive manner, we monitored the bioluminescence emissions from MIP-Luc-VU mice after adenovirus-mediated gene transfer (Figure 1A). We transferred a 3-gene combination of Pdx1, Neurod, and Mafa into the MIP-Luc-VU mice with different infectious titers to detect the bioluminescence signal 3 days after infection, according to a previous study (Figure 1B) [18]. After luciferin injection (5 mg/kg body weight, IP) into the gene-transferred mouse, within minutes the emission from the liver rose quickly and then gradually attenuated (Figure S1B). The 3-gene transfer resulted in a dose-dependent bioluminescence emission in the hepatic region, which was identified as originating from the liver by imaging of the extracted tissues and by immunohistochemistry using anti-luciferase antibody (Figure 1B, C, D). Most hepatic cells exhibit reporter expression, whereas immunohistochemistry using anti-insulin antibody showed very few luciferase-positive cells expressing insulin protein, indicating the existence of some regulatory mechanisms of the transcription and the protein levels of insulin, such as mRNA and protein degradation and leakage of produced insulin into the blood circulation (Figure S1C).


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)

Bioluminescence emission from the hepatic region of MIP-Luc-VU mice after β cell-related gene transfer.(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.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0113022-g001: Bioluminescence emission from the hepatic region of MIP-Luc-VU mice after β cell-related gene transfer.(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.
Mentions: To screen the transcriptional activity of the intrahepatic insulin gene in a noninvasive manner, we monitored the bioluminescence emissions from MIP-Luc-VU mice after adenovirus-mediated gene transfer (Figure 1A). We transferred a 3-gene combination of Pdx1, Neurod, and Mafa into the MIP-Luc-VU mice with different infectious titers to detect the bioluminescence signal 3 days after infection, according to a previous study (Figure 1B) [18]. After luciferin injection (5 mg/kg body weight, IP) into the gene-transferred mouse, within minutes the emission from the liver rose quickly and then gradually attenuated (Figure S1B). The 3-gene transfer resulted in a dose-dependent bioluminescence emission in the hepatic region, which was identified as originating from the liver by imaging of the extracted tissues and by immunohistochemistry using anti-luciferase antibody (Figure 1B, C, D). Most hepatic cells exhibit reporter expression, whereas immunohistochemistry using anti-insulin antibody showed very few luciferase-positive cells expressing insulin protein, indicating the existence of some regulatory mechanisms of the transcription and the protein levels of insulin, such as mRNA and protein degradation and leakage of produced insulin into the blood circulation (Figure S1C).

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